Unraveling the influence of CRISPR/Cas13a reaction components on enhancing trans-cleavage activity for ultrasensitive on-chip RNA detection.

The CRISPR/Cas13 nucleases have been widely documented for nucleic acid detection. Understanding the intricacies of CRISPR/Cas13's reaction components is pivotal for harnessing its full potential for biosensing applications. Herein, we report on the influence of CRISPR/Cas13a reaction components on its trans-cleavage activity and the development of an on-chip total internal reflection fluorescence microscopy (TIRFM)-powered RNA sensing system. We used SARS-CoV-2 synthetic RNA and pseudovirus as a model system. Our results show that optimizing Mg2+ concentration, reporter length, and crRNA combination significantly improves the detection sensitivity. Under optimized conditions, we detected 100 fM unamplified SARS-CoV-2 synthetic RNA using a microtiter plate reader. To further improve sensitivity and provide a new amplification-free RNA sensing toolbox, we developed a TIRFM-based amplification-free RNA sensing system. We were able to detect RNA down to 100 aM. Furthermore, the TIRM-based detection system developed in this study is 1000-fold more sensitive than the off-coverslip assay. The possible clinical applicability of the system was demonstrated by detecting SARS-CoV-2 pseudovirus RNA. Our proposed sensing system has the potential to detect any target RNA with slight modifications to the existing setup, providing a universal RNA detection platform.

Cancer Therapy Empowered by Extracellular Vesicle-Mediated Targeted Delivery.

Extracellular vesicles (EVs) are a class of nanoparticles that mediate signaling molecules delivery between donor and recipient cells. Heterogeneity in the content of EVs and their membrane surface proteins determines their unique targetability. Their low immunogenicity, capability to cross various biological barriers, and superior biocompatibility enable engineering-modified EVs to be ideal drug delivery carriers. In addition, the engineered EVs that emerge in recent years have become a powerful tool for cancer treatment through the selective delivery of bioactive molecules to therapeutic targets, such as tumor cells and stroma. Our review focuses on the various types of EV modifications and their promoting therapeutic capabilities, which provide an innovative means for cancer precision therapy.

Efficacy of concurrent chemoradiotherapy combined with nimotuzumab in the treatment of nasopharyngeal carcinoma with cervical lymph node metastasis.

OBJECTIVES: First, we retrospectively compared the clinical efficacy of concurrent chemoradiotherapy combined with nimotuzumab vs. chemoradiotherapy alone in patients with nasopharyngeal carcinoma (NPC) and cervical lymph node metastasis. Second, we analyzed the value of Ki-67 as a predictor of nimotuzumab efficacy. METHODS: From January 2012 to December 2019, 1250 patients with cervical lymph node metastasis eligible for enrollment were included, of whom 383 were treated with concurrent chemoradiotherapy combined with nimotuzumab (targeted therapy group), and 867 were treated with concurrent chemoradiotherapy (CRT group). A total of 381 pairs of patients were matched using 1:1 propensity score matching, and differences in clinical prognosis were compared between the two groups. RESULTS: Overall survival (OS) (P = 0.028), disease-free survival (DFS) (P = 0.040), and distant metastasis-free survival (DMFS) (P = 0.040) were better in the targeted therapy compared to the CRT group. Multivariate analysis revealed that clinical staging, chemotherapy, and nimotuzumab therapy were predictors of OS and DFS. In the targeted therapy group, patients with ≥ 50% Ki-67 positivity had better OS and DFS rates than those with < 50% Ki-67 positivity. CONCLUSIONS: In patients with stage N1-3 NPC and lymph node metastasis, the addition of nimotuzumab to concurrent chemoradiotherapy may provide additional survival benefits. Ki-67 is a potential biomarker with clinical predictive value for the efficacy of nimotuzumab combined with chemoradiotherapy.

The miRNA-185-5p/STIM1 Axis Regulates the Invasiveness of Nasopharyngeal Carcinoma Cell Lines by Modulating EGFR Activation-Stimulated Switch from E- to N-Cadherin.

Distant metastasis remains the primary cause of treatment failure and suggests a poor prognosis in nasopharyngeal carcinoma (NPC). Epithelial-mesenchymal transition (EMT) is a critical cellular process for initiating a tumor invasion and remote metastasis. Our previous study showed that the blockage of the stromal interaction molecule 1 (STIM1)-mediated Ca2+ signaling blunts the Epstein-Barr virus (EBV)-promoted cell migration and inhibits the dissemination and lymphatic metastasis of NPC cells. However, the upstream signaling pathway that regulates the STIM1 expression remains unknown. In this follow-up study, we demonstrated that the miRNA-185-5p/STIM1 axis is implicated in the regulation of the metastatic potential of 5-8F cells, a highly invasive NPC cell line. We demonstrate that the knockdown of STIM1 attenuates the migration ability of 5-8F cells by inhibiting the epidermal growth factor receptor (EGFR) phosphorylation-induced switch from E- to N-cadherin in vitro. In addition, the STIM1 knockdown inhibited the locoregional lymphatic invasion of the 5-8F cells in mice. Furthermore, we identified miRNA-185-5p as an upstream regulator that negatively regulates the expression of STIM1. Our findings suggest that the miRNA-185-5p/STIM1 axis regulates the invasiveness of NPC cell lines by affecting the EGFR activation-modulated cell adhesiveness. The miRNA-185-5p/STIM1 axis may serve as a potentially effective therapeutic target for the treatment of NPC.

STAT3 regulates SRGN and promotes metastasis of nasopharyngeal carcinoma through the FoxO1-miR-148a-5p-CREB1 axis.

Nasopharyngeal carcinoma (NPC), which is marked by a distinct distribution, is a common subtype of epithelial carcinoma arising from the nasopharyngeal mucosal lining. SRGN acts as an important and poor prognostic factor of NPC through multiple different mechanisms. However, the biological role and mechanism of SRGN in NPC remain unknown. Expression levels of miR-148a-5p, CREB1, FoxO1, and SRGN in NPC tissues and cell lines were tested by qRT-PCR or/and Western blot. The impacts of miR-148a-5p, CREB1, FoxO1, and SRGN on NPC cell viability, proliferation, migration, and invasion were estimated in vitro by CCK-8, colony formation, wound healing and Transwell experiments, and in vivo by a xenograft tumor model. JASPAR analysis was used to predict the binding activity of Foxo1 (CREB1) with the miR-148a-5p (SRGN) promoter, and the interaction was validated by EMSA and ChIP assays. The miR-148a-5p-CREB1 interaction was validated by a dual-luciferase reporter and RIP assays. CREB1 and SRGN were increased while miR-148a-5p was decreased in NPC. Silencing of SRGN and CREB1, as well as miR-148a-5p overexpression, repressed NPC tumor progression in vitro and in vivo. CREB1 promoted SRGN expression in NPC by targeting the promoter area of SRGN. Silencing of FoxO1 facilitated NPC tumor progression, while silencing of STAT3 repressed NPC tumor progression. FoxO1 bound to and regulated miR-148a-5p in NPC, and miR-148a-5p targeted CREB1. Additionally, FoxO1 knockdown abolished the downregulation of CREB1 and SRGN induced by STAT3 silencing. Our results suggest that STAT3 regulates SRGN and promotes the growth and metastasis of NPC through the FoxO1-miR-148a-5p-CREB1 axis.

Integrated analysis of a competing endogenous RNA network reveals key long noncoding RNAs as potential prognostic biomarkers for hepatocellular carcinoma.

Growing evidence has revealed that long noncoding RNAs (lncRNAs) have an important impact on tumorigenesis and tumor progression via a mechanism involving competing endogenous RNAs (ceRNAs). However, their use in predicting the survival of a patient with hepatocellular carcinoma (HCC) remains unclear. The aim of this study was to develop a novel lncRNA expression-based risk score system to accurately predict the survival of patients with HCC. In our study, using expression profiles downloaded from The Cancer Genome Atlas database, the differentially expressed messenger RNAs (mRNAs), lncRNAs, and microRNAs (miRNAs) were explored in patients with HCC and normal liver tissues, and then a ceRNA network constructed. A risk score system was established between lncRNA expression of the ceRNA network and overall survival (OS) or recurrence-free survival (RFS); it was further analyzed for associations with the clinical features of patients with HCC. In HCC, 473 differentially expressed lncRNAs, 63 differentially expressed miRNAs, and 1417 differentially expressed mRNAs were detected. The ceRNA network comprised 41 lncRNA nodes, 12 miRNA nodes, 24 mRNA nodes, and 172 edges. The lncRNA expression-based risk score system for OS was constructed based on six lncRNAs (MYLK-AS1, AL359878.1, PART1, TSPEAR-AS1, C10orf91, and LINC00501), while the risk score system for RFS was based on four lncRNAs (WARS2-IT1, AL359878.1, AL357060.1, and PART1). Univariate and multivariate Cox analyses showed the risk score systems for OS or RFS were significant independent factors adjusted for clinical factors. Receiver operating characteristic curve analysis showed the area under the curve for the risk score system was 0.704 for OS, and 0.71 for RFS. Our result revealed a lncRNA expression-based risk score system for OS or RFS can effectively predict the survival of patients with HCC and aid in good clinical decision-making.

Calcium Release from Endoplasmic Reticulum Involves Calmodulin-Mediated NADPH Oxidase-Derived Reactive Oxygen Species Production in Endothelial Cells.

BACKGROUND: Previous studies demonstrated that calcium/calmodulin (Ca2+/CaM) activates nicotinamide adenine dinucleotide phosphate oxidases (NOX). In endothelial cells, the elevation of intracellular Ca2+ level consists of two components: Ca2+ mobilization from the endoplasmic reticulum (ER) and the subsequent store-operated Ca2+ entry. However, little is known about which component of Ca2+ increase is required to activate NOX in endothelial cells. Here, we investigated the mechanism that regulates NOX-derived reactive oxygen species (ROS) production via a Ca2+/CaM-dependent pathway. METHODS: We measured ROS production using a fluorescent indicator in endothelial cells and performed phosphorylation assays. RESULTS: Bradykinin (BK) increased NOX-derived cytosolic ROS. When cells were exposed to BK with either a nominal Ca2+-free or 1 mM of extracellular Ca2+ concentration modified Tyrode's solution, no difference in BK-induced ROS production was observed; however, chelating of cytosolic Ca2+ by BAPTA/AM or the depletion of ER Ca2+ contents by thapsigargin eliminated BK-induced ROS production. BK-induced ROS production was inhibited by a CaM inhibitor; however, a Ca2+/CaM-dependent protein kinase II (CaMKII) inhibitor did not affect BK-induced ROS production. Furthermore, BK stimulation did not increase phosphorylation of NOX2, NOX4, and NOX5. CONCLUSIONS: BK-induced NOX-derived ROS production was mediated via a Ca2+/CaM-dependent pathway; however, it was independent from NOX phosphorylation. This was strictly regulated by ER Ca2+ contents.

Interactions between bradykinin and plasmin in the endothelial Ca2+ response.

Plasmin is a fibrinolytic factor and a serine protease that activates protease-activated receptors (PARs) to produce endothelium-derived relaxing factors such as nitric oxide and prostacyclin. Nitric oxide and prostacyclin production is regulated, at least in part, by the intracellular Ca2+ concentration in various blood vessel types. Bradykinin and plasmin stimulate vascular endothelial cells and work simultaneously in pathophysiological conditions such as thrombosis and inflammation. Here, we explored the interactions between bradykinin and plasmin in the endothelial Ca2+ response using the fluorescent indicator, Fura-2/AM, in primary cultures of porcine aortic endothelial cells (PAECs). Plasmin (0.15-15 µg/ml) and bradykinin (0.1-10 nM) increased intracellular Ca2+ concentrations in PAECs in a dose-dependent manner, and the plasmin-induced endothelial Ca2+ response occurred only once. Bradykinin (0.1-10 nM) inhibited the plasmin-induced endothelial Ca2+ response in a dose-dependent manner, however, plasmin did not affect the bradykinin-induced endothelial Ca2+ response. Pretreatment with gabexate mesilate (GM, 100 µM), a serine protease inhibitor, that blocks plasmin's proteolytic activity, fully suppressed the plasmin-induced Ca2+ response. After washout of GM and the first plasmin, the second administration of plasmin caused Ca2+ increases. However, when the first plasmin-induced Ca2+ response was blocked by pretreatment with bradykinin, the second plasmin (15 µg/ml) application did not cause any Ca2+ response, even 30 min after the washout of the first plasmin and bradykinin. Our data suggested that bradykinin regulated the plasmin-induced endothelial Ca2+ response by inhibiting the pathway downstream of the PARs' N-terminus cleavage.

Nicorandil stimulates a Na⁺/Ca²âº exchanger by activating guanylate cyclase in guinea pig cardiac myocytes.

Nicorandil, a hybrid of an ATP-sensitive K(+) (KATP) channel opener and a nitrate generator, is used clinically for the treatment of angina pectoris. This agent has been reported to exert antiarrhythmic actions by abolishing both triggered activity and spontaneous automaticity in an in vitro study. It is well known that delayed afterdepolarizations (DADs) are caused by the Na(+)/Ca(2+) exchange current (I NCX). In this study, we investigated the effect of nicorandil on the cardiac Na(+)/Ca(2+) exchanger (NCX1). We used the whole-cell patch clamp technique and the Fura-2/AM (Ca(2+) indicator) method to investigate the effect of nicorandil on I NCX in isolated guinea pig ventricular myocytes and CCL39 fibroblast cells transfected with dog heart NCX1. Nicorandil enhanced I NCX in a concentration-dependent manner. The EC50 (half-maximum concentration for enhancement of the drug) values were 15.0 and 8.7 µM for the outward and inward components of I NCX, respectively. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), a membrane-permeable analog of guanosine 3',5'-cyclic monophosphate (cGMP), enhanced I NCX. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor (10 µM), completely abolished the nicorandil-induced I NCX increase. Nicorandil increased I NCX in CCL39 cells expressing wild-type NCX1 but did not affect mutant NCX1 without a long intracellular loop between transmembrane segments (TMSs) 5 and 6. Nicorandil at 100 µM abolished DADs induced by electrical stimulation with ouabain. Nicorandil enhanced the function of NCX1 via guanylate cyclase and thus may accelerate Ca(2+) exit via NCX1. This may partially contribute to the cardioprotection by nicorandil in addition to shortening action potential duration (APD) by activating KATP channels.

The Safety and Immunological Effects of rAd5-EBV-LMP2 Vaccine in Nasopharyngeal Carcinoma Patients: A Phase I Clinical Trial and Two-Year Follow-Up.

Epstein-Barr virus (EBV)-encoded latent membrane protein 2 (LMP2) promotes nasopharyngeal carcinoma (NPC) progression. Previously, we reported that the dendritic cells (DCs) transfected with EBV-LMP2 recombinant serotype 5 adenoviruses (rAd5) induced anti-tumor effect by eliciting cytotoxic T lymphocytes (CTLs)-mediated immune response in vitro and the adenoviral vaccine of EBV-LMP2 (rAd5-EBV-LMP2) stimulated antigen-specific cellular immunity in mice. However, the safety and immunological effect of rAd5-EBV-LMP2 vaccine in human still remained unknown. Here we conducted a single-center, non-randomized, open-label, single-arm phase I clinical trial to clarify this unsolved issue. A total of 24 patients with regional advanced NPC were sequentially enrolled into three dose level groups (2×10(9), 2×10(10), 2×10(11) vp). The rAd5-EBV-LMP2 vaccines were intramuscularly injected for four times within 28 d (D0, D7, D14, D28). Blood samples were harvested immediately before every vaccination, one week and one month after the last vaccination (D0, D7, D14, D28, D35, D58). All the vaccine inoculation-related toxicities presented as grade I/II adverse events. The most frequent systemic adverse reactions were fatigue (33.0%, 8/24), myalgia (29.2%, 7/24) and cough (29.2%, 7/24), while the most common regional adverse reaction was tenderness in the inoculation site (54.2%, 13/24). In addition, proportion of CD(3+)CD(4+) cells in peripheral blood was significantly increased in the high dose group (2×10(11) vp). The rAd5-EBV-LMP2 vaccine was generally well-tolerated and the high dose (2×10(11) vp) is recommended to be adopted in phase II studies. The long-term outcome of rAd5-EBV-LMP2 vaccine inoculation is required to be determined in following placebo-controlled trials.

Bibliometric analysis reveals the research hotspots and trends of nasopharyngeal carcinoma immunotherapy.

Immunotherapy is a promising strategy for nasopharyngeal carcinoma (NPC), a common and aggressive malignancy with poor prognosis. However, the literature lacks a comprehensive and objective overview of the current research status and trends of immunotherapy-related fields in NPC. We performed a bibliometric analysis of 513 original articles and reviews in English on immunotherapy for NPC from the Web of Science Core Collection database, using CiteSpace and Bibliometrix software tools. We visualized the development trend of publications, the distribution of countries/regions, the co-occurrence of keywords, the collaboration and citation of authors, the citation of journals, the evolution of topics, and the thematic map. We found that the publication volume increased sharply after 2017, with China as the main contributor and leader, the US as an important partner, and the Netherlands as a potential innovator. The research focused on immune checkpoint inhibitors and cell therapy, which were also the hotspots of clinical trials. Tumor microenvironment, immune infiltration, multicenter studies, and novel immunotherapy were the frontier topics and the key challenges for future research. CD137l-DC, lymphoma, and chimeric antigen receptor were emerging topics with good prospects. Our study provides a valuable insight into the research status and trends of immunotherapy for NPC, which may guide future research directions and clinical applications.

[Radiation-induced sarcoma in patients with nasopharyngeal carcinoma: a single-institution retrospective study].

Objective:To analyze the clinical features, treatment methods and prognosis of radiation-induced sarcoma（RIS） of the head and neck after radiotherapy for nasopharyngeal carcinoma（NPC）, and explore its treatment strategies. Methods:A retrospective analysis was conducted on RIS patients after radiotherapy for NPC in the People's Hospital of Guangxi Zhuang Autonomous Region from January 2013 to October 2022. The time of onset, lesion location, pathological subtypes, imaging features and treatment outcomes were described, and the median survival time was statistically analyzed through follow-up. Results:This study included 10 patients with an interval of 2-27 years between NPC and RIS. The nasopharynx was the more common site of RIS, and osteosarcoma was the main pathological type. The median overall survival was 18 months. The median survival was 40 months in the surgery combined with the chemotherapy group, and 12 months in the surgery alone group. The 1-and 2-year cumulative survival rates were 48% and 36%, respectively. Prognostic analysis showed that gender, age of onset, time of sarcoma onset after radiotherapy and treatment methods might not be influencing factors for prognosis, and osteosarcomas presented a poorer prognosis than other pathological types. Conclusion:RIS is one of the most severe long-term adverse effects in patients with NPC. The prognosis of RIS is poor, and complete surgical resection of the tumor can improve patient survival rates. In cases where complete surgical resection is challenging, radiotherapy or chemotherapy may offer some improvement in tumor control.

[Effect of endoscopic surgery combined with chemotherapy and radiotherapy on prognosis of early nasopharyngeal carcinoma patients in high incidence area].

Objective:To investigate the differences in the therapeutic effects of endoscopic surgery combined with chemotherapy and endoscopic surgery combined with radiotherapy in the treatment of early nasopharyngeal carcinoma, and to select individualized treatment strategy for early nasopharyngeal carcinoma. Methods:The clinical data of 68 patients with early nasopharyngeal carcinoma（T1-2N0M0） who received surgical treatment in a high-incidence area were retrospectively analyzed. According to different treatment methods, they were divided into the surgery + chemotherapy group（n=34, treated with endoscopic surgery combined with chemotherapy） and the surgery + radiotherapy group（n=34, treated with endoscopic surgery combined with radiotherapy）. Propensity score matching was used to match the patient data between the two groups at a 1∶1 ratio. Patients were followed up, and the survival rates and hematological toxicities were compared between the two groups. Results:Twenty-four cases in the surgery + chemotherapy group and 24 cases in the surgery + radiotherapy group were successfully matched. After matching, there was no statistically significant difference in T stage, and clinical stage between the two groups（all P>0.05）. The 3-year OS and DFS in the surgery + chemotherapy group were 100.0% and 95.8%, respectively, while the 3-year OS and DFS in the surgery + radiotherapy group were 100.0% and 100.0%, respectively, with no significant difference in survival rates between the two groups（both P>0.05）. After treatment, there was no statistically significant difference in bone marrow suppression between the surgery + chemotherapy group and the surgery + radiotherapy group （all P> 0.05） Conclusion:Endoscopic surgery combined with chemotherapy and surgery combined with radiotherapy have comparable clinical efficacy in the treatment of early nasopharyngeal carcinoma, but without radiotherapy-related complications, which is worth further investigation.

Linoleic acid attenuates endothelium-derived relaxing factor production by suppressing cAMP-hydrolyzing phosphodiesterase activity.

BACKGROUND: Linoleic acid (LA) promotes monocyte chemotaxis and cell adhesion molecules such as MCP-1 and VCAM-1, which contribute to atherosclerogenesis. These molecules are restrained by endothelium-derived relaxing factors (EDRFs), such as nitric oxide (NO) and prostaglandin I2 (PGI2). Hence, the expressions of MCP-1 and VCAM-1 upregulated by LA may be partly attributable to decreased EDRF production. However, effect of LA on EDRF production remains controversial. METHODS AND RESULTS: The present study aimed to examine the effects of LA and other free fatty acids on EDRF production and the endothelial Ca(2+) responses that mediate EDRF production, using primary cultured porcine aortic endothelial cells (PAECs). LA at 0.1-5 µmol/L attenuated bradykinin (BK)-induced NO and PGI2 production while suppressing the BK-induced Ca(2+) response dose-dependently. The inhibitory effect of LA on the Ca(2+) response was eliminated by adenylate cyclase inhibitor SQ22536, boosted by cAMP-hydrolyzing phosphodiesterase (PDE) inhibitor, rolipram, and mimicked by plasma membrane permeable 8-bromo-cAMP. Moreover, LA was confirmed to dose-dependently increase intracellular cAMP levels and selectively inhibit cAMP-hydrolyzing PDE activity in vitro. In contrast, none of palmitic, stearic, or oleic acid affected BK-induced EDRF production or Ca(2+) responses, or induced intracellular cAMP accumulation. CONCLUSIONS: LA induced intracellular cAMP accumulation by inhibiting cAMP-hydrolyzing PDE activity, thus resulting in attenuation of Ca(2+) responses and EDRF production in PAECs.

Short-term hyperthermia promotes the sensitivity of MCF-7 human breast cancer cells to paclitaxel.

As a physical adjuvant approach in the treatment of solid tumors, regional hyperthermia plays a synergistic role in enhancing the efficacy of simultaneous chemotherapy. Paclitaxel (PTX) is an anti-mitotic taxane drug that is widely used in chemotherapy for the treatment of various human malignancies such as lung, ovarian, breast, and head and neck cancers. Since the possibility that hyperthermia can enhance the anti-tumor effects of PTX has not yet been investigated, the present study was designed to evaluate the effects of short-term hyperthermia on PTX-induced antitumor activity in the human breast cancer line MCF-7. It was found that short-term hyperthermia promoted PTX-induced suppression of cell proliferation. The IC for PTX was reduced from 18.2±1.0 to 15.0±0.45 nM (p<0.05). The level of PTX-induced cell apoptosis was increased from 8.5±1.2 to 16.4±2.4% (p<0.05) and from 15.2±1.4 to 34.9±2.8% (p<0.05), at the end of the first and second hyperthermia cycles, respectively; both the activity and expression of caspase-7 were enhanced. In addition, PTX-induced cell cycle arrest in the G2/M phase was further promoted by short-term hyperthermia, from 9.3±0.7 to 12.5±0.9% (p<0.05). In contrast, short-term hyperthermia affected neither tumor cell migration nor invasion in the presence or absence of PTX. The presented data thus suggest that short-term hyperthermia may serve as a feasible approach in the promotion of breast cancer cell sensitivity to PTX.

[Diagnosis of nasopharyngeal carcinoma with convolutional neural network on narrowband imaging].

Objective:To evaluate the diagnostic accuracy of the convolutional neural network（CNN） in diagnosing nasopharyngeal carcinoma using endoscopic narrowband imaging. Methods:A total of 834 cases with nasopharyngeal lesions were collected from the People's Hospital of Guangxi Zhuang Autonomous Region between 2014 and 2016. We trained the DenseNet201 model to classify the endoscopic images, evaluated its performance using the test dataset, and compared the results with those of two independent endoscopic experts. Results:The area under the ROC curve of the CNN in diagnosing nasopharyngeal carcinoma was 0.98. The sensitivity and specificity of the CNN were 91.90% and 94.69%, respectively. The sensitivity of the two expert-based assessment was 92.08% and 91.06%, respectively, and the specificity was 95.58% and 92.79%, respectively. There was no significant difference between the diagnostic accuracy of CNN and the expert-based assessment （P=0.282, P=0.085）. Moreover, there was no significant difference in the accuracy in discriminating early-stage and late-stage nasopharyngeal carcinoma（P=0.382）. The CNN model could rapidly distinguish nasopharyngeal carcinoma from benign lesions, with an image recognition time of 0.1 s/piece. Conclusion:The CNN model can quickly distinguish nasopharyngeal carcinoma from benign nasopharyngeal lesions, which can aid endoscopists in diagnosing nasopharyngeal lesions and reduce the rate of nasopharyngeal biopsy.

STIM1-regulated exosomal EBV-LMP1 empowers endothelial cells with an aggressive phenotype by activating the Akt/ERK pathway in nasopharyngeal carcinoma.

BACKGROUND: Stromal interaction molecule 1 (STIM1)-mediated Ca2+ signaling regulates tumor angiogenesis in nasopharyngeal carcinoma (NPC), an Epstein-Barr virus (EBV)-related human malignancy. However, the mechanism by which STIM1 modulates endothelial functional phenotypes contributing to tumor angiogenesis remains elusive. METHODS: NPC cell-derived exosomes were isolated via differential centrifugation and observed using transmission electron microscopy. Exosome particle sizes were assessed by nanoparticle tracking analysis (NTA). Uptake of exosomes by recipient ECs was detected by fluorescent labeling of the exosomes with PKH26. Tumor angiogenesis-associated profiles were characterized by determining cell proliferation, migration, tubulogenesis and permeability in human umbilical vein endothelial cells (HUVECs). Activation of the Akt/ERK pathway was assessed by detecting the phosphorylation levels using Western blotting. A chick embryo chorioallantoic membrane (CAM) xenograft model was employed to study tumor-associated neovascularization in vivo. RESULTS: We found that NPC cell-derived exosomes harboring EBV-encoded latent membrane protein 1 (LMP1) promoted proliferation, migration, tubulogenesis and permeability by activating the Akt/ERK pathway in ECs. STIM1 silencing reduced LMP1 enrichment in NPC cell-derived exosomes, thereby reversing its pro-oncogenic effects in an Akt/ERK pathway-dependent manner. Furthermore, STIM1 knockdown in NPC cells blunted tumor-induced vascular network formation and inhibited intra-tumor neovascularization in the chorioallantoic membrane (CAM) xenograft model. CONCLUSION: STIM1 regulates tumor angiogenesis by controlling exosomal EBV-LMP1 delivery to ECs in the NPC tumor microenvironment. Blocking exosome-mediated cell-to-cell horizontal transfer of EBV-associated oncogenic signaling molecules may be an effective therapeutic strategy for NPC.

Detection of Frog Virus 3 by Integrating RPA-CRISPR/Cas12a-SPM with Deep Learning.

A fast, easy-to-implement, highly sensitive, and point-of-care (POC) detection system for frog virus 3 (FV3) is proposed. Combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a, a limit of detection (LoD) of 100 aM (60.2 copies/µL) is achieved by optimizing RPA primers and CRISPR RNAs (crRNAs). For POC detection, smartphone microscopy is implemented, and an LoD of 10 aM is achieved in 40 min. The proposed system detects four positive animal-derived samples with a quantitation cycle (Cq) value of quantitative PCR (qPCR) in the range of 13 to 32. In addition, deep learning models are deployed for binary classification (positive or negative samples) and multiclass classification (different concentrations of FV3 and negative samples), achieving 100 and 98.75% accuracy, respectively. Without temperature regulation and expensive equipment, the proposed RPA-CRISPR/Cas12a combined with smartphone readouts and artificial-intelligence-assisted classification showcases the great potential for FV3 detection, specifically POC detection of DNA virus.

Fluid dynamics of the upper airway in pediatric patients with severe laryngomalacia.

Laryngomalacia is the top cause of pediatric laryngeal wheeze. We used computational fluid dynamics to study the inspiratory airflow dynamics in severe pediatric laryngomalacia. Computed tomography was performed on the upper airways of two infants, one with severe laryngomalacia and one with normal airway, and 3D models were reconstructed. ANSYS CFD-POST software was used to simulate airflow in these models to compare the volumetric flow rate, flow velocity, pressure, wall shear, and vortex. The volume flow rate in the laryngomalacia model was significantly reduced compared with the control model. Under inspiratory pressures, the peak flow velocity, pressure, and shear force in the control model appeared at the soft palate stenosis, while that in the laryngomalacia model appeared at the supraglottis stenosis. In both models, the maximum flow velocity and shear force increased with decreasing inspiratory pressure, while the minimum pressure decreased with decreasing inspiratory pressure. In the control model, the airflow vortex appeared anteriorly below the posterior section of the soft palate. In the laryngomalacia model, the vortex appeared anteriorly below the posterior section of the soft palate and anteriorly below the vocal folds. Our methodology provides a new mechanistic understanding of pediatric laryngomalacia.

Exosome-mediated remodeling of the tumor microenvironment: From local to distant intercellular communication.

Extracellular vesicles (EVs) are membrane-enveloped nanoscale particles that carry various bioactive signaling molecules secreted by cells. Their biological roles depend on the original cell type from which they are derived and their inclusions. Exosomes, a class of EVs, are released by almost all eukaryotic cell types, including tumor cells. Tumor cell-derived exosomes mediate signal transduction between tumor cells and surrounding non-tumor cells. This intercellular communication actively contributes to the remodeling of the tumor microenvironment (TME) to enable tumor growth, invasion, and metastasis. This review summarizes the latest progress in the exploration of exosome-mediated cell-cell communication implicated in TME remodeling and underlying mechanisms. We focus on the role of cell-cell interactions mediated by tumor cell-derived exosomes in promoting invasion and metastasis, and their potential as a therapeutic intervention target against distant metastasis. We also discuss the clinical translational significance of tumor-derived exosomes for early diagnosis, efficacy and progression evaluations.