Abstract Immunotherapy is increasingly being utilized in the management of thymic epithelial tumors (TET). High expression levels of programmed death receptor 1 (PD-1) and its ligand 1 (PD-L1) have been observed in TET, suggesting their potential as prognostic indicators for disease progression and the effectiveness of immunotherapy in TET. We propose that the utilization of quantitative imaging biomarkers could potentially serve as an alternative surrogate for predicting the PD-L1 expression status in clinical decision-making assistance. A total of 124 patients with pathologically confirmed TET (57 PD-L1 positive, 67 PD-L1 negative) were retrospectively enrolled and allocated into training and validation cohorts in a ratio of 7:3. Radiomics features were extracted from T1-weighted, T2-weighted fat suppression, and apparent diffusion coefficient (ADC) map images to establish a radiomics signature in the training cohort. Multivariate logistic regression analysis was conducted to develop a combined radiomics nomogram that incorporated clinical, conventional MR features, or ADC model for evaluation purposes. The performance of each model was compared using receiver operating characteristics analysis, while discrimination, calibration, and clinical efficiency of the combined radiomics nomogram were assessed. The radiomics signature, consisting of four features, demonstrated a favorable ability to predict and differentiate between PD-L1 positive and negative TET patients. The combined radiomics nomogram, which incorporates the peri-cardial invasion sign, ADC value, WHO classification, and radiomics signature, showed excellent performance (training cohort: area under the curve [AUC] = 0.903; validation cohorts: AUC = 0.894). The calibration curve and decision curve analysis further confirmed the clinical usefulness of this combined model. The decision curve analysis demonstrated the clinical utility of the integrated radiomics nomogram. The radiomics signature serves as a valuable tool for predicting the PD-L1 status of TET patients. Furthermore, the integration of radiomics nomogram enhances the personalized prediction capability.

Extrachromosomal circular DNA (eccDNA) was discovered several decades ago, but little is known about its function. With the development of sequencing technology, several library preparation methods have been developed to elucidate the biogenesis and function of eccDNA. However, different treatment methods have certain biases that can lead to their erroneous interpretation. To address these issues, we compared the performance of different library preparation methods. Our investigation revealed that the utilization of rolling-circle amplification (RCA) and restriction enzyme linearization of mitochondrial DNA (mtDNA) significantly enhanced the efficiency of enriching extrachromosomal circular DNA (eccDNA). However, it also introduced certain biases, such as an unclear peak in ∼160-200 bp periodicity and the absence of a typical motif pattern. Furthermore, given that RCA can lead to a disproportionate change in copy numbers, eccDNA quantification using split and discordant reads should be avoided. Analysis of the genomic and elements distribution of the overall population of eccDNA molecules revealed a high correlation between the replicates, and provided a possible stability signature for eccDNA, which could potentially reflect different cell lines or cell states. However, we found only a few eccDNA with identical junction sites in each replicate, showing a high degree of heterogeneity of eccDNA. The emergence of different motif patterns flanking junctional sites in eccDNAs of varying sizes suggests the involvement of multiple potential mechanisms in eccDNA generation. This study comprehensively compares and discusses various essential approaches for eccDNA library preparation, offering valuable insights and practical advice to researchers involved in characterizing eccDNA.

ObjectivesTo validate an appropriate evaluation method of liver fibrosis assessment based on the unique pathological features of biliary atresia (BA) that could well predict its prognosis.MethodsA total of 68 patients with BA who underwent Kasai procedure (KP) and an intraoperative liver biopsy, followed up from January 2019 to December 2021, were recruited in a retrospective analysis. Ishak, Metavir, and BA-specific staging systems in relation to outcomes were analyzed using logistic regression, COX proportional hazard regression, Kaplan-Meier analysis, etc.ResultsKaplan-Meier analysis determined a significant difference in native liver survival according to the BA-specific stage (p = 0.002). The ROC curve analysis for predicting prognosis showed that the AUC of BA-specific staging combined with iBALF and severe bile duct proliferation (BDP) (0.811, 95% CI: 0.710–0.913, p < 0.0001) was higher than BA-specific staging alone (0.755, 95% CI: 0.639–0.872, p < 0.001).ConclusionsThe BA-specific staging system reflects the condition of the liver fibrosis, and its combination with iBALF and severe BDP helps to better evaluate the prognosis of patients with BA.

Objective: To investigate the oncological outcomes of active surveillance (AS) in patients showing thyroid nodules measuring≤1 cm with highly suspicious ultrasound features. Methods: A prospective single-center cohort study. A total of 534 patients with highly suspicious thyroid nodules (2015 American Thyroid Association Nodule Sonographic Patterns and Risk of Malignancy: High Suspicion) were enrolled in this study, the patients received AS at Peking Union Medical College Hospital between January 2017 and November 2022 to assess oncological outcomes (disease progression, recurrence/metastasis rate, etc). The patients were followed up every 6 months for physical examination and neck ultrasound examination. And the value of tumor volume changes in evaluating tumor enlargement was explored too. Results: There were 413 females and 121 males in this cohort, with a mean age of (42.6±11.8) years. During a median follow-up period of 45.6 months (ranged from 3.5 to 176.0 months), disease progression occurred in 26 patients (4.9%) with highly suspicious thyroid nodules, characterized by a minimum 3-mm increase in tumor diameter in 19 patients (3.6%) and lymph node metastases in 7 patients (1.3%). Forty-seven (8.8%) patients opted for delayed surgery, with 29 patients due to a change in preference. There was no significant differences in pathologic and follow-up outcomes between patients with disease progression and preference change. Patients aged≤40 years had a higher cumulative incidence of 5-year disease progression than those aged>40 years (4.9% vs 1.9%, P=0.060). No patients experienced distant metastases or deaths. Among the 595 high-risk thyroid nodules with continuous volume assessment results and an increase in nodule diameter of less than 3 mm (including all high-risk nodules in patients with single or multiple nodules), 184 (30.9%) and 79 (13.3%) nodules exhibited volume increases of more than 50% and 100%, respectively, in multiple measurements. Among the nodules with volume changes exceeding 50% and 100%, the proportion of nodules with a baseline tumor diameter of≤0.5 cm was significantly higher than those with a diameter of>0.5 cm, at 69.0% vs 31.0% (P<0.001) and 77.2% vs 22.8% (P<0.001), respectively. Conclusions: Active surveillance in patients with highly suspicious subcentimeter thyroid nodules has good short-term oncological outcomes and can be considered a safe alternative to surgery. Due to the large variability in the measurement results of tumor volume, it is not suitable as an indicator for evaluating tumor enlargement.

Hepatocellular carcinoma (HCC) is among the most prevalent and lethal cancers worldwide. The tumor microenvironment (TME) contributes to the poor response of patients with HCC to current therapies, while tumor vascular endothelial cells (ECs) are fundamental TME components that significantly contribute to tumor progression. However, the specific functions and mechanisms of tumor vascular ECs in HCC remain unclear. We screened and validated diacylglycerol kinase gamma (DGKG) hyper-expression specifically in HCC tumor vascular ECs. Single-cell RNA-sequencing, cytometry by time-of-flight, and invitro and invivo studies were performed to investigate the functions of endothelial DGKG. Multiplexed immunohistochemistry staining and flow cytometry were used to evaluate changes in the TME. Functionally, endothelial DGKG promotes tumor angiogenesis and immunosuppressive regulatory T-cell differentiation in HCC. Of significance, we found that HIF-1α activates DGKG transcription by directly binding to its promoter region under hypoxia. Upregulated DGKG promotes HCC progression by recruiting ubiquitin specific peptidase 16 to facilitate ZEB2 deubiquitination, which increases TGF-β1 secretion, thus inducing tumor angiogenesis and regulatory T-cell differentiation. Importantly, targeting endothelial DGKG potentiated the efficiency of dual blockade of PD-1 and VEGFR-2. Hypoxia-induced EC-specific DGKG hyper-expression promotes tumor angiogenesis and immune evasion via the ZEB2/TGF-β1 axis, suggesting EC-specific DGKG as a potential therapeutic target for HCC. Here, we reported that hypoxia-induced endothelial cell-specific DGKG hyper-expression promotes angiogenesis and immune evasion in HCC by recruiting USP16 for K48-linked deubiquitination and inducing the subsequent stabilization of ZEB2, leading to increased TGF-β1 secretion. Most importantly, endothelial DGKG inhibition greatly improved the efficacy of the dual combination of anti-VEGFR2 and anti-PD-1 treatment in a mouse HCC model, significantly inhibiting the malignant progression of HCC and improving survival. This preclinical study supports the targeting of endothelial DGKG as a potential strategy for precision HCC treatment.

The current study aimed to investigate the use of fractional anisotropy (FA) and mean diffusivity (MD) as a non-invasive probe for assessing chemoradiotherapy (CRT) efficacy in 32 high-grade glioma patients (HGGs). Diffusion tensor imaging (DTI) was performed pre- and post-CRT, and FA and MD maps were constructed with Philips MRI 1.5 T beside Intellispace software. Tumor regression and progression were assessed by changes in tumor size monitored with T1-contrast enhancement, with 15 cases (46.9%) demonstrating regression and 17 cases (53.1%) showing progression. The cut-off values of mean pre- CRT FA and MD in the tumor core were 0.345 and 0.957 × 10–3 mm2/s, respectively, with a sensitivity of 73.3% and 86.7% and a specificity of 70.6% and 52.9%. In the peritumoral zone, the mean values of pre-FA and MD were 0.415 and 1.21 × 10–3 mm2/s, respectively, with a sensitivity of 93.3% and 86.7% and a specificity of 76.5% for both. Comparatively, the cut-off values of mean after CRT FA and MD in the tumor core were 0.295 and 0.965 × 10–3 mm2/s, respectively, with a sensitivity of 99.2% and 98.1% and a specificity of 94.1% and 93.1%. Higher post-CRT FA and MD values were associated with better treatment response rates, indicating that FA and MD are valuable DTI metrics for assessing CRT efficacy in HGG regression and progression without invasive procedures.

Abstract Introduction: HIF-1 α is an oxygen concentration-sensitive protein that regulates the emergency response to hypoxia. Microglial pyroptosis is closely related to cerebral ischemic injury. In this study, through in vitro cell modeling in order to elucidate the regulatory mechanism of HIF-1 α on microglial pyroptosis and inflammation in the acute phase of the cerebral ischemia and hypoxia. Methods: In vitro experiments with microglia, the acute phase oxyoxygenation model (oxygen-glucose deprivation / reoxygenation, OGD/R) was established, and the optimal time of intervention was determined by measuring the survival rate with CCK-8. Overexpression and suppressed expression of HIF-1α factors, and the experiment were divided into blank group (Group A), OGD/R model group (Group B), OGD/R model + FG-4592 intervention group (Group C),model + siRNA negative control group (Group D), and model + HIF-1 α -siRNA group (Group E). Cell proliferation and cell supernatant LDH concentration were measured in different groups. IL-1β and IL-18 levels in cell supernatants were determined by ELISA. Western blot The protein expression levels of HIF-1α, GSDMD-D, GSDMD-N, cle-Caspase-1, and NLRP3 were determined by the same method. Results: Finally, hypoxia for 6 h reoxygenation for 12 h was the optimal intervention time. Compared with groups B and D, group C increased cell proliferation and decrease in LDH, IL-1β and IL-18 was statistically significant (P&lt;0.05), while group Edecreased cell proliferation and increase in LDH, IL-1β and IL-18 concentrations were statistically significant (P&lt;0.05). Compared with groups B and D, GSDMD-D, GSDMD-N, clean-Caspase-1, and NLRP 3 proteins were significantly lower in group C but significantly increased in group E cells (P &lt;0.05). Conclusion: BV2 cells in the acute phase of cerebral ischemia and hypoxia, found that upregulated HIF-1α expression would reduce microglial pyroptosis and modulation of inflammatory responses. Furthermore, we found that HIF-1α inhibited microglial pyroptosis by inhibiting the NLRP3/GSDMD pathway, and this conclusion provides a potential target for the clinical treatment of ischemic stroke in the acute phase.

Abstract Profilin 2 (PFN2) is an actin-binding protein important for cancer initiation and progression. However, the function and precise role of PFN2 in non-small cell lung cancer (NSCLC) remain unclear. Here, we assessed the expression levels of PFN2 in tissue from patients with NSCLC; identified binding partners using mass spectrometry, co-immunoprecipitations, and molecular modeling; and studied the angiogenic-promoting function of PFN2 using 3D droplet cultures and tube-formation assays. We found that upregulated PFN2 expression is associated with poor prognosis in patients with NSCLC. Knockdown of PFN2 significantly impaired the proliferation and angiogenesis of NSCLC cells, both in vivo and in vitro. Mechanistically, PFN2 physically interacts with pyruvate kinase M2 (PKM2) and modulates extracellular-signal regulated kinase 1/2 (ERK1/2)-mediated phosphorylation of PKM2 at S37, which substantially increases its nuclear translocation. PFN2 knockdown lead to reduced expression of PKM2, p65-NF-κB, and hypoxia-inducible factor (HIF)-1α and inhibited the nuclear translocation of PKM2. This resulted in impaired formation of the PKM2-HIF-1α-p65-NF-κB transcription complex, leading to decreased expression of its downstream target genes VEGFA, CCND1, and MAP2K5. Moreover, mutations in PFN2 at Y134 or S138 impaired its binding to PKM2, nuclear translocation of PKM2, and functionally inhibited angiogenesis in lung cancer cells. Overall, our data provide insights into the role of PFN2 in the regulation of lung cancer angiogenesis and indicate that PFN2 may serve as a therapeutic target against NSCLC.

Gatrodin is an active compound with cardio-protective potentials. In the current study, the mechanism underlying the effects of gastrodin was explored. Myocardial infarction was induced and handled with gastrodin. The role of miR-30a-5p and its downstream effector, ATG5, in the cardio-protective effects of gastrodin was verified. Gastrodin induced the dys-expresseion of 72 miRs in oxygen/glucose deprivation (OGD) cardiomyocytes and miR-30a-5p was selected as the potential therapeutic target. Gastrodin increased viability and suppressed autophagy in OGD cardiomyocytes, which was associated with the induction of miR-30a-5p and deactivation of ATG5. In rat models, gastrodin attenuated structure deterioration and dysfunction in heart, and suppressed autophagy. At molecular level, miR-30a-5p was induced and ATG5 were down-regulated. After the suppression of miR-30a-5p or the overexpression of ATG5, the cardio-protective effects of gastrodin were compromised. Gastrodin induced the expression of miR-30a-5p, which contributed to the attenuation of autophagy in infarcted heart tissues.