ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97.

The t(X,17) chromosomal translocation, generating the ASPSCR1::TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCCs), frustrating efforts to identify therapeutic targets for these rare cancers. Here, proteomic analysis identifies VCP/p97, an AAA+ ATPase with known segregase function, as strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1::TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1::TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributes with ASPSCR1::TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrate the oncogenic transcriptional signature of ASPSCR1::TFE3, by facilitating assembly of higher-order chromatin conformation structures demonstrated by HiChIP. Finally, ASPSCR1::TFE3 and VCP demonstrate co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP's potential as a novel therapeutic target.

Whether the use of intravenous contrast-enhanced ultrasound can improve the accuracy of T stage diagnosis of gastric cancer?

OBJECTIVE: To explore whether the use of intravenous contrast-enhanced ultrasound (CEUS) can improve the diagnostic accuracy of T stage of gastric cancer on the basis of oral contrast-enhanced ultrasound (OCEUS). METHODS: Eighty-one patients with gastric cancer who underwent preoperative CEUS. Criteria for T staging was AJCC Stage 8th Edition. To compare the diagnostic accuracy, sensitivity and specificity of OCEUS and CEUS for T staging of gastric cancer using pathological results as the gold standard. RESULTS: There were differences in the diagnostic accuracy of T2 (P = 0.048), T3(P = 0.002) and T4 (P = 0.000) between OCEUS and CEUS. And there was no difference in diagnostic accuracy for T1 (P = 0.118). CEUS significantly improved diagnostic sensitivity and specificity in T3 (42.86% to 61.90% for sensitivity, 86.96% to 93.75% for specificity), and T4 (64.71% to 76.47% for sensitivity, 78.33% to 95.74% for specificity). CONCLUSION: Compared with OCEUS, CEUS did improve the diagnostic accuracy of T2, T3, and T4 stages of gastric cancer.

Long scleral tunnel technique for prevention of drainage tube-related complications during Ahmed glaucoma valve implantation.

To evaluate the safety and efficacy of modified Ahmed glaucoma valve (AGV) implantation (long scleral tunnel technique) in the treatment of neovascularization glaucoma (NVG). This retrospective observational case series included 23 patients (23 eyes) diagnosed with NVG secondary to proliferative diabetic retinopathy from January 2020 to June 2021. All 23 cases received anti-vascular endothelial growth factor treatment. Then, after 3 to 7 days, these cases were treated with modified AGV implantation (long scleral tunnel technique) and were followed up for at least 6 months. The best corrected visual acuity, intraocular pressure, numbers of antiglaucoma medications used, and postoperative complications were observed at 1 week and 1, 3, and 6 months after treatment. With treatment, the mean best corrected visual acuity improved significantly (P < .001) from 1.62 ±â€…0.52 logMAR preoperatively to 1.29 ±â€…0.36 logMAR at the 6-month follow-up. The mean postoperative intraocular pressure was significantly lower than that before modified AGV implantation during follow-up period, decreasing from 45.48 ±â€…7.86 mm Hg preoperatively to 14.87 ±â€…1.96 mm Hg at 1 week, 18.39 ±â€…2.25 mm Hg at 1 month, 16.61 ±â€…1.47 mm Hg at 3 months, and 17.48 ±â€…1.38 mm Hg at 6 months (F = 256.646, P < .001). The median number of antiglaucoma medications used by patients also significantly decreased from 3 (3-4) preoperatively to 0 (0-1) at the 6-month follow-up after surgery (Z = -4.248, P < .001). Postoperative complications included hyphema in 2 cases and vitreous hemorrhage in 1 case, and all 3 patients achieved satisfactory recovery with treatment. No drainage tube-related complications occurred among our patients. Long scleral tunnel technique is a safe and effective surgical treatment for NVG with fewer drainage tube-related complications.

Discovery of novel 2,3,4,5-tetrahydrospiro[benzo[c]azepine-1,1'-cyclohexan]-5-ol derivatives as PARP-1 inhibitors.

As an essential marker of cancer treatment, PARP-1 inhibitors could effectively kill tumor cells through a mechanism known as synthetic lethality and are used to treat a variety of cancers. In order to explore novel PARP-1 inhibitors, a series of 22 novel erythrina derivatives were reported and preliminarily explored their mechanism of action. The antitumor activities against four human cancer cell lines including A549, OVCAR-3, HCT-116, and MCF-7 were evaluated, and the preliminary SARs were summarized. Among them, compound 11b exhibited better anti-proliferative effects against A549 cells (IC50 = 1.95 µM). The SI results showed that compound 11b had low toxicity. Moreover, compound 11b displayed excellent PARP-1 inhibitory activities with IC50 values of 19.24 nM. In addition, molecular docking studies provided the rational binding modes of compound 11b in complexes with PARP-1. The flow cytometry assays revealed that compound 11b could induce apoptosis of A549 cells (P < 0.001). Simultaneously, compound 11b could effectively reduce the formation of PAR (P < 0.001). The ADMET prediction results indicated compound 11b had similar properties to rucaparib. Collectively, compound 11b has potential research value for further investigation.

ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97.

The t(X,17) chromosomal translocation, generating the ASPSCR1-TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCC), frustrating efforts to identify therapeutic targets for these rare cancers. Proteomic analysis showed that VCP/p97, an AAA+ ATPase with known segregase function, was strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1-TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1-TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributed with ASPSCR1-TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrated the oncogenic transcriptional signature of ASPSCR1-TFE3, by facilitating assembly of higher-order chromatin conformation structures as demonstrated by HiChIP. Finally, ASPSCR1-TFE3 and VCP demonstrated co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP's potential as a novel therapeutic target.

Effects of peripheral blood neutrophil/lymphocyte ratio levels and their changes on the prognosis of patients with early cervical cancer.

Although some studies have reported on the levels and clinical significance of peripheral blood neutrophil/lymphocyte ratio (NLR) in cervical cancer, the role of NLR levels and their changes preoperatively and postoperatively in early cervical cancer remain unclear. Our analyses explored the preoperative and postoperative NLR in 203 patients with stage I-IIA cervical cancer and evaluated the relationship between NLR changes, clinicopathological characteristics, and patient prognosis. The cut-off preoperative and postoperative NLR values were determined using receiver operating characteristic curve analysis. Preoperative NLR correlated with age, menopausal status, tumor size, and vascular infiltration, whereas postoperative NLR correlated with tumor differentiation. Patients with cervical cancer with a high preoperative NLR had significantly shorter overall survival (OS) and progression-free survival (PFS) than other patients, whereas PFS was significantly lower in the high postoperative NLR group. When comparing postoperative and preoperative NLR values, we observed a significantly higher rate of increase in postmenopausal patients and those without vascular infiltration than that among premenopausal patients and those with vascular infiltration. However, no clear difference in prognosis was observed between the groups with increased and decreased NLR. Therefore, a high peripheral blood NLR may predict a poor prognosis in patients with early cervical cancer. The effect of NLR changes on the prognosis of patients with cervical cancer requires further verification in multicenter studies.

Pre-operative Prediction of Invasiveness in Renal Cell Carcinoma: The Role of Conventional Ultrasound and Contrast-Enhanced Ultrasound.

OBJECTIVE: It is known that in patients with renal cell carcinoma (RCC), the invasiveness of the tumor is closely related to the treatment and prognosis. Currently, histologic diagnosis of RCC is typically established after surgical removal of tumors or after biopsy. The use of non-invasive imaging modalities to predict the invasiveness of RCC is of great clinical value, particularly before surgery. In this study, the differences in conventional ultrasound (US) and contrast-enhanced ultrasound (CEUS) features between invasive and non-invasive RCC were analyzed with the aim of providing more accurate and valuable information for diagnosis and treatment to clinically optimize the treatment plan in a non-invasive manner and improve the prognosis of patients. METHODS: Conventional US and CEUS features of 163 patients (total of 164 RCCs), obtained from the Lanzhou University Second Hospital in the period ranging from March 2021 to September 2022, were retrospectively analyzed. Patients were categorized into two groups: invasive group (n = 44) and non-invasive group (n = 120), with surgical pathology as reference standard. Receiver operating characteristic curves were drawn to evaluate the feasibility of differentiation. RESULTS: The possibility of an intrarenal lesion/kidney ratio >50% in the invasive group (13/44, 29.5%) was significantly higher than that in the non-invasive group (8/120, 6.7%) (p < 0.001). The absence of perilesional rim-like enhancement was more likely to imply invasive RCC (30/44, 68.2%) than non-invasive RCC (100/120, 83.3%) (p = 0.049) and was an independent predictor of invasive RCC. As for CEUS quantitative features, there were statistically significant differences in peak intensity (p = 0.009) or peak enhancement (p = 0.010), taking the largest range of lesion as the region of interest. CONCLUSION: Conventional US and CEUS features may help in the differentiation of invasive RCC from non-invasive RCC and have potential application value in the pre-operative prediction of RCC invasiveness.

[Comparison of HPLC fingerprints and determination of main components of Viticis Fructus from different species].

In order to comprehensively evaluate the quality of Viticis Fructus, this study established HPLC fingerprints and evaluated the quality of 24 batches of Viticis Fructus samples from different species by similarity evaluation and multivariate statistical analysis(PCA, HCA, PLS-DA). On this basis, an HPLC method was established to compare the content differences of the main components, including casticin, agnuside, homoorientin, and p-hydroxybenzoic acid. The analysis was performed on the chromatographic column(Waters Symmetry C_(18)) with a gradient mobile phase of acetonitrile(A)-0.05% phosphoric acid solution(B) at the flow rate of 1 mL·min~(-1) and detection wavelength of 258 nm. The column temperature was 30 ℃ and the injection volume was 10 µL. The HPLC fingerprint of 24 batches of Viticis Fructus samples was established with 21 common peaks, and nine peaks were identified. Similarity analysis was carried out based on chromatographic data of 24 batches of chromatographic data of Viticis Fructus, and the results showed that except for DYMJ-16, the similarity of Vitex trifolia var. simplicifolia was ≥0.900, while that of V. trifolia was ≤0.864. In addition, the similarity analysis of two different species showed that the similarity of 16 batches of V. trifolia var. simplicifolia was 0.894-0.997 and that of the eight batches of V. trifolia was between 0.990 and 0.997. The results showed that the similarity of fingerprints of these two species was different, but the similarity between the same species was good. The results of the three multivariate statistical analyses were consistent, which could distinguish the two different species. The VIP analysis results of PLS-DA showed that casticin and agnuside contributed the most to the distinction. The content determination results showed that there was no significant difference in the content of homoorientin and p-hydroxybenzoic acid in Viticis Fructus from different species, but the content of casticin and agnuside was significantly different in different species(P<0.01). The content of casticin was higher in V. trifolia var. simplicifolia, while agnuside was higher in V. trifolia. The findings of this study show that there are differences in fingerprint similarity and component content of Viticis Fructus from different species, which can provide references for the in-depth study of the quality and clinical application of Viticis Fructus.

GRK2 Kinases in the Primary Cilium Initiate SMOOTHENED-PKA Signaling in the Hedgehog Cascade.

During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO, and the ensuing PKA-C binding and inactivation, are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors.

Can the Vesical Imaging Reporting and Data System (VI-RADS) score be applied in conventional ultrasound and contrast-enhanced ultrasound to differentiate muscle-invasive bladder cancer?

AIM: To investigate the value of vesical imaging reporting and data system (VI-RADS) score based on conventional ultrasound and contrast-enhanced ultrasound (CEUS) in differentiating muscle-invasive bladder cancer (MIBC). MATERIAL AND METHODS: Thirty-eight patients who underwent conventional ultrasound and CEUS examinations at our ultrasound department between March 2021 and June 2022 were retrospectively included in this study. Two sonographers analyzed the conventional ultrasound and CEUS images of each patient separately and scored them in strict accordance with VI-RADS scoring system based on conventional ultrasound and CEUS. The VI-RADS score of each lesion was compared with the pathological findings to assess the diagnostic performance of VI-RADS. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the VI-RADS score in the diagnosis of MIBC were analyzed when the cutoff values were 3 and 4, respectively. Interobserver agreement was evaluated using the weighted-kappa coefficient (κ). RESULTS: The final pathological results evidenced 25 (65.8%) non-muscle-invasive bladder cancers (NMIBCs) and 13 (34.2%) MIBCs. When the VI-RADSscore ≥ 4, the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the VI-RADS score for predicting muscle-invasive bladder cancer were 92.3%, 84.0%, 75.0%, 95.5%, and 86.8%, respectively. The area under the receiver operating curve for the VI-RADS score was 0.882 (95%CI:0.761-1). Inter-reader agreement for VI-RADS scoreswas good. CONCLUSION: The VI-RADS score based on conventional ultrasound and CEUS is useful in differentiating muscleinvasive bladder cancer.

Diagnostic Value of Contrast-Enhanced Ultrasound Features for WHO/ISUP Grading in Renal Cell Carcinoma.

OBJECTIVES: By analyzing the differences of contrast-enhanced ultrasound (CEUS) features between low- and high-grade of WHO/ISUP grading, to explore the diagnostic value of CEUS in evaluating the prognosis of renal cell carcinoma (RCC). METHODS: The qualitative and quantitative features of CEUS in 69 patients with RCC confirmed by surgical pathology in the Lanzhou University Second Hospital from March to October 2021 were retrospectively analyzed. Patients were categorized into two groups: low-grade group (n = 22) and high-grade group (n = 47), with surgical pathology as reference standard. The diagnostic performance of statistically significant CEUS features was evaluated by receiver operating characteristic (ROC) curves. RESULTS: There were statistically significant differences in enhancement degree (P = .032) and quantitative features such as slopelesion (P = .034), the differences between lesion and cortex in arrive time (∆AT = ATlesion - ATcortex , P = .013), peak intensity(∆PI = [PIlesion - PIcortex ]/PIcortex , P = .003), area under the curve (∆Area = Arealesion - Areacortex , P = .008) in two groups, and the sensitivity was 70.2% and specificity was 71.4% of ∆PI, which has a high diagnostic performance in the differentiation of low-grade group from high-grade group (P = .005). CONCLUSIONS: CEUS features such as ∆PI, may help differentiate low-grade RCC from high-grade RCC. CEUS has a promising application prospect in preoperative evaluation of the prognosis of RCC.

Comparison of HPLC fingerprints and determination of main components of Viticis Fructus from different species / 中国中药杂志

In order to comprehensively evaluate the quality of Viticis Fructus, this study established HPLC fingerprints and evaluated the quality of 24 batches of Viticis Fructus samples from different species by similarity evaluation and multivariate statistical analysis(PCA, HCA, PLS-DA). On this basis, an HPLC method was established to compare the content differences of the main components, including casticin, agnuside, homoorientin, and p-hydroxybenzoic acid. The analysis was performed on the chromatographic column(Waters Symmetry C_(18)) with a gradient mobile phase of acetonitrile(A)-0.05% phosphoric acid solution(B) at the flow rate of 1 mL·min~(-1) and detection wavelength of 258 nm. The column temperature was 30 ℃ and the injection volume was 10 μL. The HPLC fingerprint of 24 batches of Viticis Fructus samples was established with 21 common peaks, and nine peaks were identified. Similarity analysis was carried out based on chromatographic data of 24 batches of chromatographic data of Viticis Fructus, and the results showed that except for DYMJ-16, the similarity of Vitex trifolia var. simplicifolia was ≥0.900, while that of V. trifolia was ≤0.864. In addition, the similarity analysis of two different species showed that the similarity of 16 batches of V. trifolia var. simplicifolia was 0.894-0.997 and that of the eight batches of V. trifolia was between 0.990 and 0.997. The results showed that the similarity of fingerprints of these two species was different, but the similarity between the same species was good. The results of the three multivariate statistical analyses were consistent, which could distinguish the two different species. The VIP analysis results of PLS-DA showed that casticin and agnuside contributed the most to the distinction. The content determination results showed that there was no significant difference in the content of homoorientin and p-hydroxybenzoic acid in Viticis Fructus from different species, but the content of casticin and agnuside was significantly different in different species(P<0.01). The content of casticin was higher in V. trifolia var. simplicifolia, while agnuside was higher in V. trifolia. The findings of this study show that there are differences in fingerprint similarity and component content of Viticis Fructus from different species, which can provide references for the in-depth study of the quality and clinical application of Viticis Fructus.

Repeated stellate ganglion blockade for the treatment of ventricular tachycardia storm in patients with nonischemic cardiomyopathy: a new therapeutic option for patients with malignant arrhythmias / 中华心血管病杂志

Objectives: This study sought to describe our institutional experience of repeated percutaneous stellate ganglion blockade (R-SGB) as a treatment option for drug-refractory electrical storm in patients with nonischemic cardiomyopathy (NICM). Methods: This prospective observational study included 8 consecutive NICM patients who had drug-refractory electrical storm and underwent R-SGB between June 1, 2021 and January 31, 2022. Lidocaine (5 ml, 1%) was injected in the vicinity of the left stellate ganglion under the guidance of ultrasound, once per day for 7 days. Data including clinical characteristics, immediate and long-term outcomes, and procedure related complications were collected. Results: The mean age was (51.5±13.6) years. All patients were male. 5 patients were diagnosed as dilated cardiomyopathy, 2 patients as arrhythmogenic right ventricular cardiomyopathy and 1 patient as hypertrophic cardiomyopathy. The left ventricular ejection fraction was 37.8%±6.6%. After the treatment of R-SGB, 6 (75%) patients were free of electrical storm. 24 hours Holter monitoring showed significant reduction in ventricular tachycardia (VT) episodes from 43.0 (13.3, 276.3) to 1.0 (0.3, 34.0) on the first day following R-SGB (P<0.05) and 0.5 (0.0, 19.3) after whole R-SGB process (P<0.05). There were no procedure-related major complications. The mean follow-up was (4.8±1.1) months, and the median time of recurrent VT was 2 months. Conclusion: Minimally invasive R-SGB is a safe and effective method to treat electrical storm in patients with NICM.

A PKA inhibitor motif within SMOOTHENED controls Hedgehog signal transduction.

The Hedgehog (Hh) cascade is central to development, tissue homeostasis and cancer. A pivotal step in Hh signal transduction is the activation of glioma-associated (GLI) transcription factors by the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO). How SMO activates GLI remains unclear. Here we show that SMO uses a decoy substrate sequence to physically block the active site of the cAMP-dependent protein kinase (PKA) catalytic subunit (PKA-C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation-induced inhibition. Using a combination of in vitro, cellular and organismal models, we demonstrate that interfering with SMO-PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism uncovered echoes one used by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly. More broadly, our findings define a mode of GPCR-PKA communication that may be harnessed by a range of membrane receptors and kinases.

Application value of shear-wave elastography combined with monochrome superb microvascular imaging in renal allograft chronic rejection.

BACKGROUND: Conventional ultrasound (US), which include gray scale US and Doppler US, is the first-line imaging modality for the evaluation of renal allograft; however, conventional US indicators have limitations. OBJECTIVE: To explore the application value of shear-wave elastography (SWE) combined with monochrome superb microvascular imaging (mSMI) in renal allograft chronic rejection (CR). METHODS: From November 2021 to February 2022 in the Lanzhou University Second Hospital, the US features of 54 patients with renal allograft were retrospectively analyzed. Patients were categorized into two groups: stable group(n = 44) and CR group(n = 10), with clinical diagnosis as reference standard. The vascular index (VI) on mSMI and parenchymal stiffness were measured in the middle cortex of all renal allografts and receiver operating characteristic (ROC) curves were drawn to evaluate the feasibility of differentiation. Statistically significant US features and biochemical indicators such as creatinine were scored, and the results of the scores were analyzed by ROC curve. RESULTS: The VI on mSMI of the stable group (49.5±2.0) was significantly greater than that of the CR group (33.8±5.9) (P = 0.028). There was a statistically significant difference in parenchymal stiffness between stable group (16.2kPa±1.2) and CR group (33.9kPa±6.6) (P = 0.027). The sensitivity was 90% and specificity was 81.8% of the scores in the differentiation of stable group from CR group (cut-off value, 2; P = 0.000). CONCLUSION: SWE combined with mSMI may help differentiate stable renal allograft from renal allograft CR and have the potential application value in the diagnosis of renal allograft CR.

Synthesis and evaluation of novel HER-2 inhibitors to exert anti-breast cancer ability through epithelial-mesenchymal transition (EMT) pathway.

Human epidermal growth factor receptor 2 (HER-2) is an essential member of the receptor tyrosine kinase (RTK) superfamily and has been reported as a critical method for treating HER-2 positive breast cancer. Here, we retained (E)-4-methyl-2-(4-(trifluoromethyl)styryl)oxazole, a fragment of HER-2 inhibitor Mubritinib, and synthesized 32 novel compounds from it. We screened out the most potential compound Q7j with HER-2 positive breast cancer cells through MTT assays, which possessed low toxicity on normal cells (MCF7-10A). Subsequently, wound healing, transwell, western blotting, and immunofluorescence experiments were performed, and it was found that compound Q7j could suppress cell migration by inhibiting the phosphorylation of HER-2 and affecting the expression of EMT-related proteins. Moreover, the SKBR3 orthotopic xenograft model confirmed that compound Q7j was more effective than Mubritinib in inhibiting the proliferation of cancer cells. In general, compound Q7j was a potential HER-2 inhibitor in treating breast cancer, which may be of great significance for developing and improving HER-2 small molecule inhibitors.

A chalcone-syringaldehyde hybrid inhibits triple-negative breast cancer cell proliferation and migration by inhibiting CKAP2-mediated FAK and STAT3 phosphorylation.

BACKGROUND: Although triple-negative breast cancer (TNBC) accounts for only 15% of breast cancer cases, it is associated with a high relapse rate and poor outcome after standard treatment. Currently, the effective drugs and treatment strategies for TNBC remain limited, and thus, developing effective treatments for TNBC is pressing. Several studies have demonstrated that both chalcone and syringaldehyde have anticancer effect, but their potential anti-TNBC bioactivity are still unknown. PURPOSE: The present study aimed to synthesize a chalcone-syringaldehyde hybrid (CSH1) and explore its potential anti-TNBC effects and the underlying molecular mechanism. METHODS: Cell cytotoxicity was determined by 3-(4,5-dimethythiazol)-2,5-diphenyltetrazolium bromide (MTT). The activity of cell proliferation was measured by colony formation assay and 5-ethynyl-2'-deoxyuridine (EdU) staining assay. Cell cycle distribution and cell apoptosis were determined by fluorescence-activated cell sorter (FACS). The situation of DNA damage was observed using fluorescence microscopy. The ability of cell-matrix adhesion, migration and invasion was detected using cell adhesion assay and transwell assay. Transcriptome sequencing was performed to find out the changed genes. Levels of various signaling proteins were assessed by western blotting. RESULTS: CSH1 treatment triggered DNA damage and inhibited DNA replication, cell cycle arrest, and cell apoptosis via suppressing signal transducer and activator of transcription 3 (STAT3) phosphorylation. Whole genome RNA-seq analysis suggested that 4% of changed genes were correlated to DNA damage and repair, and nearly 18% of changed genes were functionally related to cell adhesion and migration. Experimental evidence indicated that CSH1 treatment significantly affected the distribution of focal adhesion kinase (FAK) and its phosphorylation, resulting in cell-matrix-adhesion reduction and migration inhibition of TNBC cells. Further mechanistic studies indicated that CSH1 inhibited TNBC cell proliferation, adhesion, and migration by inhibiting cytoskeleton-associated protein 2 (CKAP2)-mediated FAK and STAT3 phosphorylation signaling. CONCLUSION: These results suggest that CKAP2-mediated FAK and STAT3 phosphorylation signaling is a valuable target for TNBC treatment, and these findings also reveal the potential of CSH1 as a prospective TNBC drug.

Development and Validation of a Nomogram for Predicting Overall Survival in Patients with Second Primary Small Cell Lung Cancer After Non-Small Cell Lung Cancer: A SEER-Based Study.

Background: Non-small cell lung cancer (NSCLC) survivors are at an increased risk of developing second primary malignancies, such as small cell lung cancer. This paper sought to establish a prognostic nomogram to assess overall survival (OS) in patients with second primary small cell lung cancer (SPSCLC) after NSCLC. Methods: 420 patients who developed SPSCLC after NSCLC were randomly split into the training and validation groups. A nomogram was established by stepwise regression. Area under the curve (AUC) and calibration plots were applied to assess the prognostic performance of the nomogram. Concordance index (C-index), integrated discrimination improvement (IDI), net reclassification index (NRI) and decision curve analysis (DCA) were performed to compare the nomogram with the American Joint Committee on Cancer (AJCC) 8th staging system. Survival risk classification was constructed based on the nomogram. Results: Five variables were chosen to construct the nomogram. The AUC showed that it had a satisfactory discrimination ability. All calibration plots displayed good concordance between nomogram and observation. The C-index, IDI, NRI and DCA showed the nomogram was superior to the AJCC 8th staging system. The Kaplan-Meier curves suggested huge differences in prognosis among the three risk groups. Conclusion: This study build a nomogram and risk stratification system for predicting probabilities of OS in patients with SPSCLC after NSCLC, which can help clinicians in individualized survival assessment and treatment decisions.

Identification of cross-reactive CD8+ T cell receptors with high functional avidity to a SARS-CoV-2 immunodominant epitope and its natural mutant variants.

Despite the growing knowledge of T cell responses in COVID-19 patients, there is a lack of detailed characterizations for T cell-antigen interactions and T cell functions. Here, with a predicted peptide library from SARS-CoV-2 S and N proteins, we found that specific CD8+ T cell responses were identified in over 75% of COVID-19 convalescent patients (15/20) and an epitope from the N protein, N361-369 (KTFPPTEPK), was the most dominant epitope from our selected peptide library. Importantly, we discovered 2 N361-369-specific T cell receptors (TCRs) with high functional avidity that were independent of the CD8 co-receptor. These TCRs exhibited complementary cross-reactivity to several presently reported N361-369 mutant variants, as to the wild-type epitope. Further, the natural functions of these TCRs in the cytotoxic immunity against SARS-CoV-2 were determined with dendritic cells (DCs) and the lung organoid model. We found that the N361-369 epitope could be normally processed and endogenously presented by these different types of antigen presenting cells, to elicit successful activation and effective cytotoxicity of CD8+ T cells ex vivo. Our study evidenced potential mechanisms of cellular immunity to SARS-CoV-2, and illuminated potential ways of viral clearance in COVID-19 patients. These results indicate that utilizing CD8-independent TCRs against SARS-CoV-2-associated antigens may provide functional superiority that is beneficial for the adoptive cell immunotherapies based on natural or genetically engineered T cells. Additionally, this information is highly relevant for the development of the next-generation vaccines with protections against continuously emerged SARS-CoV-2 mutant strains.

Clinical characteristics and long-term follow-up results of radiofrequency ablation for the treatment of ventricular tachycardia in patients with arrhythmogenic left ventricular cardiomyopathy / 中华心血管病杂志

Objective: To investigate the acute and long-term outcome of catheter ablation for the treatment of ventricular tachycardia (VT) in patients with arrhythmogenic left ventricular cardiomyopathy (ALVC). Methods: This retrospective, cross-sectional study enrolled ALVC patients undergoing radiofrequency ablation for the treatment of VT at the First Affiliated Hospital of Nanjing Medical University from January 2011 to December 2018 and collected their clinical characteristics and intraoperative electrophysiological examination. Patients were followed up every 6 months after radiofrequency ablation until August 2021. Echocardiographic results and VT recurrence post radiofrequency ablation were analysed. Results: Totally 12 patients were enrolled (mean age: (42±15) years, 11 males(11/12)). The mean of left ventricular end diastolic diameter (LVDd) and left ventricular ejection fraction (LVEF) were (51±5)mm and (65±5)%, respectively. Twelve VTs were induced in 10 patients during the electrophysiological study, and the mean tachycardia cycle length was (293±65) ms. Three-dimensional substrate mapping revealed the diseased area at endocardial site in one patient, at epicardial sites in the other 11 patients (involved endocardial sites in 2 cases) with the basal part near the mitral annulus being the predilection for the substrate (10/11). After the catheter ablation at the endocardial and epicardial sites respectively, the complete procedure endpoint was achieved in all patients (VT cannot be induced post ablation). The median follow-up time was 65 (25, 123) months. One patient was lost to follow-up, and the other 11 patients survived without VT. No significant cardiac function deterioration was detected by the echocardiographic examination ((51±5)mm vs. (52±5)mm, P>0.05 for LVDd, (65±5)% vs. (60±6)%, P>0.05 for LVEF) at the end of follow-up. Conclusion: After radiofrequency ablation, the complete procedure endpoint is achieved in ALVC patients, and the catheter ablation provides long-term ventricular tachycardia control during the long-term follow-up.