Enhanced Calcium Signal Induces NK Cell Degranulation but Inhibits Its Cytotoxic Activity.

Although the mechanism of NK cell activation is still unclear, the strict calcium dependence remains the hallmark for lytic granule secretion. A plethora of studies claiming that impaired Ca2+ signaling leads to severely defective cytotoxic granule exocytosis accompanied by weak target cell lysis has been published. However, there has been little discussion about the effect of induced calcium signal on NK cell cytotoxicity. In our study, we observed that small-molecule inhibitor UNC1999, which suppresses global H3K27 trimethylation (H3K27me3) of human NK cells, induced a PKD2-dependent calcium signal. Enhanced calcium entry led to unbalanced vesicle release, which resulted into fewer target cells acquiring lytic granules and subsequently being killed. Further analyses revealed that the ability of conjugate formation, lytic synapse formation, and granule polarization were normal in NK cells treated with UNC1999. Cumulatively, these data indicated that induced calcium signal exclusively enhances unbalanced degranulation that further inhibits their cytotoxic activity in human NK cells.

Development and validation of a selenium metabolism regulators associated prognostic model for hepatocellular carcinoma.

BACKGROUND: Selenium metabolism has been implicated in human health. This study aimed to identify a selenium metabolism regulator-based prognostic signature for hepatocellular carcinoma (HCC) and validate the role of INMT in HCC. METHODS: Transcriptome sequencing data and clinical information related to selenium metabolism regulators in TCGA liver cancer dataset were analysed. Next, a selenium metabolism model was constructed by multiple machine learning algorithms, including univariate, least absolute shrinkage and selection operator, and multivariate Cox regression analyses. Then, the potential of this model for predicting the immune landscape of different risk groups was evaluated. Finally, INMT expression was examined in different datasets. After knockdown of INMT, cell proliferation and colony formation assays were conducted. RESULTS: A selenium metabolism model containing INMT and SEPSECS was established and shown to be an independent predictor of prognosis. The survival time of low-risk patients was significantly longer than that of high-risk patients. These two groups had different immune environments. In different datasets, including TCGA, GEO, and our PUMCH dataset, INMT was significantly downregulated in HCC tissues. Moreover, knockdown of INMT significantly promoted HCC cell proliferation. CONCLUSIONS: The current study established a risk signature of selenium metabolism regulators for predicting the prognosis of HCC patients. INMT was identified as a biomarker for poor prognosis of HCC.

Evaluation of a high-sensitivity SARS-CoV-2 antigen test on the fully automated light-initiated chemiluminescent immunoassay platform.

OBJECTIVES: To describe a high-sensitivity SARS-CoV-2 antigen test that is based on the fully automated light-initiated chemiluminescent immunoassay (LiCA®), and to validate its analytical characteristics and clinical agreement on detecting SARS-CoV-2 infection against the reference molecular test. METHODS: Analytical performance was validated and detection limits were determined using different types of nucleocapsid protein samples. 798-pair anterior nasal swab specimens were collected from hospitalized patients and asymptomatic screening individuals. Agreement between LiCA® antigen and real-time reverse transcription polymerase chain reaction (rRT-PCR) was evaluated. RESULTS: Repeatability and within-lab precision were 1.6-2.3%. The C5∼C95 interval was -5.1-4.6% away from C50. Detection limits in average (SD) were 325 (±141) U/mL on the national reference panel, 0.07 (±0.04) TCID50/mL on active viral cultures, 0.27 (±0.09) pg/mL on recombinant nucleocapsid proteins and 1.07 (±1.01) TCID50/mL on inactivated viral suspensions, respectively. LiCA detected a median of 374-fold (IQR 137-643) lower levels of the viral antigen than comparative rapid tests. As reference to the rRT-PCR method, overall sensitivity and specificity were determined to be 97.5% (91.4-99.7%) and 99.9% (99.2-100%), respectively. Total agreement between both methods was 99.6% (98.7-99.9%) with Cohen's kappa 0.98 (0.96-1). A positive detection rate of 100% (95.4-100%) was obtained as Ct≤37.8. CONCLUSIONS: The LiCA® system provides an exceptionally high-sensitivity and fully automated platform for the detection of the SARS-CoV-2 antigen in nasal swabs. The assay may have high potential use for large-scale population screening and surveillance of COVID-19 as an alternative to the rRT-PCR test.

The integrate profiling of single-cell and spatial transcriptome RNA-seq reveals tumor heterogeneity, therapeutic targets, and prognostic subtypes in ccRCC.

Clear-cell renal cell carcinoma (ccRCC) is the most common type of RCC; however, the intratumoral heterogeneity in ccRCC remains unclear. We first identified markers and biological features of each cell cluster using bioinformatics analysis based on single-cell and spatial transcriptome RNA-sequencing data. We found that gene copy number loss on chromosome 3p and amplification on chromosome 5q were common features in ccRCC cells. Meanwhile, NNMT and HILPDA, which are associated with the response to hypoxia and metabolism, are potential therapeutic targets for ccRCC. In addition, CD8+ exhausted T cells (LAG3+ HAVCR2+), CD8+ proliferated T cells (STMN+), and M2-like macrophages (CD68+ CD163+ APOC1+), which are closely associated with immunosuppression, played vital roles in ccRCC occurrence and development. These results were further verified by whole exome sequencing, cell line and xenograft experiments, and immunofluorescence staining. Finally, we divide patients with ccRCC into three subtypes using unsupervised cluster analysis. and generated a classifier to reproduce these subtypes using the eXtreme Gradient Boosting algorithm. Our classifier can help clinicians evaluate prognosis and design personalized treatment strategies for ccRCC. In summary, our work provides a new perspective for understanding tumor heterogeneity and will aid in the design of antitumor therapeutic strategies for ccRCC.

Electrochemical oxidation diminished toxicity of zearalenone significantly, while reduction increased.

Zearalenone (ZEN), one of the most common mycotoxins in cereals, poses a severe health risk to humans. In this study, electrochemical oxidation and reduction degraded ZEN in solution completely within 8 min and 20 min. The structure of ZEN products was elucidated by mass spectrometry (MS), and their toxicity was evaluated by ECOSAR software and cytotoxicity assay. From simulation, electrochemical oxidation products had lower acute and chronic toxicity, and the product at 9.0 V is not harmful (LC50/EC50 greater than 100 mg/L, ChV greater than 10 mg/L). CCK-8 assay further confirmed their less cytotoxicity. To our surprise, LC50, EC50, and ChVs of all electrochemical reduction products were lower than 1 mg/L, and cell viabilities were less than ZEN, meaning the higher toxicity of electrochemical reduction products. On this Basis, electrochemical oxidation was applied in ZEN contaminated wheat with a degradation rate of 92.32 ± 2.37%, indicating its potential to degrade ZEN practically.

Prevention, Diagnosis and Eradication of Mycoplasma Contamination in Cell Culture.

Mycoplasmas, which are the smallest and simplest prokaryotes, lack a cell wall but possess the ability to undergo self-replication. Mycoplasma contamination is a common problem for laboratories engaging in cell culture. Due to their small size, Mycoplasmas can easily permeate filters designed to prevent bacterial and fungal contamination in cell culture. Although Mycoplasma contamination usually does not result in cell death, it can significantly affect cell proliferation, metabolism, and cause chromosomal aberrations. Therefore, it is crucial to detect and eliminate Mycoplasma contamination in cell culture. This step-by-step protocol presents a comprehensive approach to prevent Mycoplasma contamination in cell culture, as well as to detect and eradicate Mycoplasma to ensure accurate experimental and sequencing results.

Lsd1 safeguards T-cell development via suppressing endogenous retroelements and interferon responses.

The histone demethylase Lsd1 has been shown to play multiple essential roles in mammalian biology. However, its physiological functions in thymocyte development remain elusive. We observed that the specific deletion of Lsd1 in thymocytes caused significant thymic atrophy and reduced peripheral T cell populations with impaired proliferation capacity. Single-cell RNA sequencing combined with strand-specific total RNA-seq and ChIP-seq analysis revealed that ablation of Lsd1 led to the aberrant derepression of endogenous retroelements, which resulted in a viral mimicry state and activated the interferon pathway. Furthermore, the deletion of Lsd1 blocked the programmed sequential down-regulation of CD8 expression at the DP→CD4+CD8lo stage and induced an innate memory phenotype in both thymic and peripheral T cells. Single-cell TCR sequencing revealed the kinetics of TCR recombination in the mouse thymus. However, the preactivation state after Lsd1 deletion neither disturbed the timeline of TCR rearrangement nor reshaped the TCR repertoire of SP cells. Overall, our study provides new insight into the function of Lsd1 as an important maintainer of endogenous retroelement homeostasis in early T-cell development.

NTF2 Upregulation in HNSCC: a Predictive Marker and Potential Therapeutic Target Associated With Immune Infiltration.

Background: Head and neck squamous cell carcinoma (HNSCC) is a type of malignant tumor with an increasing incidence worldwide and a meager 5-year survival rate. It is known that nuclear transporter factor 2 (NTF2) transports related proteins into the nucleus physiologically. However, the role of NTF2 in HNSCC remains unclear. Methods: In this study, RNA-Seq data of HNSCC samples with corresponding clinical information were obtained from The Cancer Genome Atlas (TCGA) database. In addition, other expression profiling data were downloaded from the Gene Expression Omnibus (GEO) database. The differential expressions of NTF2, along with the overall survival (OS) rates were identified and analyzed. Then, the clinical features and expression levels of NTF2 were utilized to develop a prognostic model. The study also utilized the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) methods to determine the related pathways of NTF2. Furthermore, the Tumor Immune Estimation Resource (TIMER) database was referenced to discover the immune correlation of NTF2. In this research investigation, RT-qPCR, western blotting, Cell Counting Kit-8 (CCK-8) assay, wound-healing assay, and immunohistochemical (IHC) staining methods were adopted to perform experimental verifications. Results: This study's results confirmed that the NTF2 expressions were significantly increased in HNSCC tissue when compared with normal tissue. In addition, the high expression levels of NTF2 were found to be associated with poor prognoses, which was confirmed via the IHC validations of HNSCC samples with survival data. The results of functional enrichment analysis showed that the NTF2 was associated with epithelial cell growth, skin differentiation, keratosis, and estrogen metabolism. Furthermore, the expressions of NTF2 were determined to be negatively involved with immune infiltrations and correlated with immune checkpoint blockade (ICB) responses following various ICB therapy strategies. The results of the CCK-8 assay and wound-healing assay confirmed the NTF2's promoting effects on the proliferation and migration of tumor cells. Conclusions: This study defined a novel prognostic model associated with the expressions of NTF2, which was shown to be independently related to the OS of HNSCC. It was concluded in this study that NTF2 might be a potential diagnostic and prognostic biomarker for HNSCC.

Analysis of risk factors for lateral lymph node metastasis in papillary thyroid carcinoma: A retrospective cohort study.

Objective: To investigate the risk factors for lateral lymph node metastasis (LLNM) in papillary thyroid carcinoma (PTC). Methods: A retrospective analysis of 209 patients with PTC who underwent primary surgery at the Beijing Friendship Hospital affiliated with Capital Medical University from November 2014 to November 2018 was performed. The patients were divided into the LLNM group and the non-LLNM group. The clinical and pathological characteristics of the patients were analysed. The risk factors for LLNM were analysed by univariate and multivariate analyses. Results: The incidence of LLNM was 13.4% in PTC patients. Univariate analysis showed that the maximum diameter of the primary tumour > 2 cm (P < 0.001), bilateral primary tumour (P = 0.020), extrathyroidal extension (ETE) (P < 0.001), central lymph node metastasis (CLNM) (P < 0.001), and CLNM number ≥ 5 (P < 0.001) were significantly associated with LLNM. Multivariate logistic regression analysis showed that the maximum diameter of the primary tumour > 2 cm, ETE, and CLNM were independent risk factors for LLNM (OR values were 3.880, 5.202, and 4.474, respectively). There were 6 patients with skip lateral cervical lymph node metastasis, accounting for 21% of all LLNM patients. Conclusion: This study revealed several independent risk factors for predicting LLNM in PTC patients, such as the maximum diameter of the primary tumour > 2 cm, ETE and CLNM. Lateral neck dissection may be recommended in PTC patients with those risk factors. Paying attention to the occurrence of skip lateral cervical lymph node metastasis during the clinical diagnosis and treatment processes is necessary.

Single-Cell Sequencing Reveals the Novel Role of Ezh2 in NK Cell Maturation and Function.

Natural killer (NK) cells are lymphocytes primarily involved in innate immunity and exhibit important functional properties in antimicrobial and antitumoral responses. Our previous work indicated that the enhancer of zeste homolog 2 (Ezh2) is a negative regulator of early NK cell differentiation and function through trimethylation of histone H3 lysine 27 (H3K27me3). Here, we deleted Ezh2 from immature NK cells and downstream progeny to explore its role in NK cell maturation by single-cell RNA sequencing (scRNA-seq). We identified six distinct NK stages based on the transcriptional signature during NK cell maturation. Conditional deletion of Ezh2 in NK cells resulted in a maturation trajectory toward NK cell arrest in CD11b SP stage 5, which was clustered with genes related to the activating function of NK cells. Mechanistically, we speculated that Ezh2 plays a critical role in NK development by activating AP-1 family gene expression independent of PRC2 function. Our results implied a novel role for the Ezh2-AP-1-Klrg1 axis in altering the NK cell maturation trajectory and NK cell-mediated cytotoxicity.

Prognostic value of α2δ1 in hypopharyngeal carcinoma: A retrospective study.

Voltage-dependent calcium channel subunit alpha-2/delta-1 (α2δ1) has been identified as a marker of cancer stem cells in multiple malignant tumor types. However, α2δ1's role in the prognosis of hypopharyngeal squamous cell carcinoma (HSCC) was not reported. In our study, ten pairs of HSCC and peritumoral normal tissues were used for immunohistochemistry assessment. And α2δ1 expression levels of 34 more HSCC samples were also evaluated, represented by the integral optic density using Image-Pro Plus. Clinicopathological associations and prognostic value of α2δ1 were analyzed. As a result, α2δ1 expression was frequently increased in HSCC tissues. Although the correlation between patients' clinicopathological characteristics and their α2δ1 expression levels was not significant, α2δ1 expression was significantly correlated with unfavorable overall survival (OS) (P = 0.018) and progression-free survival (PFS) (P = 0.023). Univariate and multivariate cox regression analyses suggested α2δ1's prognostic role for both OS and PFS (P = 0.013 and 0.011, respectively). This study specifically demonstrated that α2δ1 regularly increased in HSCC compared with peritumoral tissues, and α2δ1 could act as a promising prognostic marker in HSCC patients.

DDAH2 suppresses RLR-MAVS-mediated innate antiviral immunity by stimulating nitric oxide-activated, Drp1-induced mitochondrial fission.

The RIG-I-like receptor (RLR) signaling pathway is pivotal for innate immunity against invading viruses, and dysregulation of this molecular cascade has been linked to various diseases. Here, we identified dimethylarginine dimethylaminohydrolase 2 (DDAH2) as a potent regulator of the RLR-mediated antiviral response in human and mouse. Overexpression of DDAH2 attenuated RLR signaling, whereas loss of DDAH2 function enhanced RLR signaling and suppressed viral replication ex vivo and in mice. Upon viral infection, DDAH2 relocated to mitochondria, where it induced the production of nitric oxide (NO) and the activation of dynamin-related protein 1 (Drp1), which promoted mitochondrial fission and blocked the activation of innate immune responses mediated by mitochondrial antiviral signaling (MAVS). TANK-binding kinase 1 (TBK1), a kinase downstream of MAVS, inhibited DDAH2 by phosphorylating DDAH2 at multiple sites. Our study thus identifies a reciprocal inhibitory loop between the DDAH2-NO cascade and the RLR signaling pathway that fine-tunes the antiviral immune response.

Histone methyltransferase Ezh2 negatively regulates NK cell terminal maturation and function.

NK cells are innate lymphoid cells that play important roles in tumor eradication and viral clearance. We previously found that deletion or inhibition of the histone methyltransferase Ezh2 (enhancer of zeste homolog 2) in hematopoietic stem and progenitor cells (HSPCs) from both mice and humans enhanced the commitment and cytotoxicity of NK cells to tumor cells. This study tested the hypothesis that inhibiting Ezh2, especially in NK lineage cells, could also affect NK cell development and function. We crossed Ezh2fl/fl mice with Ncr1iCre mice to delete the Ezh2 gene in immature NK cells and downstream progeny. Ezh2 deficiency increased the total number of NK cells and promoted NK cell terminal differentiation, as the percentages of the most mature CD27- CD11b+ subsets were increased. The NK cell cytotoxicity against tumor cells in vitro was enhanced, with increased degranulation and IFN-γ production. In addition, during the process of human NK cells differentiating from HSPCs , inhibiting EZH2 catalytic activity at day 14 (when NK lineage commitment began) also resulted in increased proportions of mature NK cells and cytotoxicity. Furthermore, RNA-seq and CUT&RUN-qPCR assays showed that the effects of Ezh2 may be based on its direct modulation of the expression of the transcription factor Pbx1 (pre-B-cell leukemia transcription factor 1), which has been reported to promote NK cell development. In summary, we demonstrate that Ezh2 is a negative regulator of NK cell terminal maturation and function.

A Novel Ferroptosis-Related Gene Model for Overall Survival Predictions of Bladder Urothelial Carcinoma Patients.

INTRODUCTION: Bladder cancer is the most common urinary tract malignancy, and 90% of bladder tumors are urothelial cell carcinomas. Ferroptosis is a new form of cell death discovered in recent years, which is an iron-dependent form of cell death characterized by the lethal intracellular accumulation of lipid-based reactive oxygen species. Ferroptosis is considered to be a double-edged sword for cancer and cancer therapy. MATERIALS AND METHODS: In the current study, expression profiles of bladder cancer (BLCA) specimens were obtained from The Cancer Genome Atlas (TCGA) RNA-Seq database. Ferroptosis-related genes were downloaded from the FerrDb website. The ferroptosis-related differentially expressed genes (DEGs) which were related to overall survival (OS) were first identified. The least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression methods were utilized to develop a ferroptosis-related prognostic model (FRPM). In addition, a nomogram model based on FRPM and clinicopathological features was successfully constructed and validated. In addition, gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and single-sample gene set enrichment analysis (ssGSEA) methods were utilized in this study in order to compare the DEGs between the high-risk and low-risk groups. This study also adopted RT-qPCR, CCK-8 assay, and scratch assay methods to perform experimental verification processes. RESULTS AND DISCUSSION: A 7-gene FRPM was constructed in this research investigation in order to stratify the patients into two groups according to their risk scores. The results of this study's survival analysis and time-dependent receiver operating characteristic (ROC) analysis demonstrated that the model had achieved a stable performance level. This multivariate Cox regression results revealed that the FRPM was an independent prognostic predictor for the OS of BLCA patients and the results were displayed using a nomogram. In addition, the ROC analysis, concordance index (C-index), calibration plots, and decision curve analysis (DCA) curves further indicated that this study's nomogram method enabled valuable prediction results. The functional enrichment analysis results suggested that the DEGs between the high- and low-risk groups played vital roles in the progression of the ferroptosis. Also, the ssGSEA indicated that the immune status was different between the two groups. This study found that the RT-qPCR results had confirmed the differential expressions of DEGs in the tissue samples, and the CCK-8 assay and scratch assay results confirmed the promoting effects of SCD on the proliferation and migration of tumor cells. CONCLUSIONS: This study defined a novel prognostic model of seven ferroptosis-related genes, which proved to be independently associated with the OS of BLCA. A nomogram method was developed for the purpose of providing further insight into the accurate predictions of BLCA prognoses.

SNF5 deficiency induces apoptosis resistance by repressing SATB1 expression in Sézary syndrome.

SNF5, is a core member of the SWI/SNF chromatin remodeling complex. It's deficiency leads to multiple types of aggressive cancer. Sézary syndrome, a leukemic variant of cutaneous T-cell lymphoma, is characterized by its resistance to apoptosis. Although the cause of apoptosis resistance is still poorly understood, recent evidence has revealed the importance of SATB1 in the apoptosis resistance of Sézary syndrome. In this study, we show that SNF5 is an upstream regulator of SATB1 in several conditions and that both are deficient in Sézary cells. Additionally, SNF5 not only controls the expression of SATB1, but also utilizes SATB1 to recruit itself to specific sites. Overexpression of SNF5 induces SATB1 expression and partially reverse apoptosis resistance phenotype in Sézary cells. These results suggest that both SNF5 and SATB1 may regulate apoptosis-related genes in Sézary syndrome. Thus, targeting SWI/SNF complex may represent a promising approach for Sézary syndrome therapy.

Chromatin state dynamics during NK cell activation.

Studies of Natural Killer (NK) cell cytotoxicity have mainly focused on the balance of activating and inhibitory receptors, signaling transduction, calcium influx, formation of immune synapse, and cytolytic degranulation. However, little is known about the chromatin state of NK cells and the impact of its changes during target recognition. In this study, we investigate the contribution of chromatin state dynamics during NK cell activation by comprehensively analyzing a set of microarray data and two sets of Chromatin Immunoprecipitation-Sequencing (ChIP-seq) data. We find that the expression of several histone demethylases and methyltransferases was influenced upon stimulation. Furthermore, we notice that a series of genes, including PI3KCA, NFATC1and TNFSF9, which play important roles during NK cell activation, were at 'poised' state prior to activation, and that modifications of H3K4me3 and H3K27me3 on these promotors were sensitive to stimulation with Phorbol Myristate Acetate (PMA) and Ionomycin (Iono) in the NK92MI cell line. Finally, we demonstrate that a series of small molecule inhibitors, which are specific to H3K4 and H3K27 modification, enhance degranulation or the expression levels of IFN-γ and TNF-α. Our results suggest that the histone modification state has a profound impact on NK cell activation, and provide novel insights into the regulation of NK cellular cytotoxicity and immunoregulatory function by chromatin state dynamics.