Heterogeneous nuclear ribonucleoparticule A1/A2 (hnRNP A1/A2) and splicing factor 2/alternative splicing factor (SF2/ASF) are pivotal for precursor messenger RNA (pre-mRNA) splicing. Interferon regulatory factor-3 (IRF-3) plays critical roles in host defense against viral and microbial infection. Truncated IRF-3 proteins resulting from alternative splicing have been identified and characterized as functional antagonists to full-length IRF-3. In this study, we examined the molecular mechanism for splicing regulation of IRF-3 pre-mRNA and first reported the regulatory effect of hnRNP A1/A2 and SF2/ASF on IRF-3 splicing and activation. RNA interference-mediated depletion of hnRNP A1/A2 or SF2/ASF in human non-small cell lung cancer (NSCLC) cells increased exclusion of exons 2 and 3 of IRF-3 gene and reduced expression levels of IRF-3 protein and IRF-3 downstream effector molecules interferon-beta and CXCL10/IP-10. In addition, direct binding of hnRNP A1 and SF2/ASF to specific binding motifs in IRF-3 intron 1 was confirmed by RNA electrophoretic mobility shift assay. Subsequent minigene splicing assay showed that IRF-3 minigenes with mutated hnRNPA 1/A2 or SF2/ASF binding motifs increased exclusion of exons 2 and 3. Moreover, knockdown of hnRNP A1/A2 or SF2/ASF in NSCLC cells reinforced phytohemagglutinin-induced tumor necrosis factor-alpha release by peripheral blood mononuclear cells (PBMC) but suppressed that of interleukin-10 in NSCLC/PBMC co-cultures. Taken together, our results suggest that specific knockdown for hnRNP A1/A2 or SF2/ASF increase exclusion of exons 2 and 3 of IRF-3 pre-mRNA and influence immunomodulatory functions of human NSCLC cells.

Background Knowledge of immune cell phenotypes, function, and developmental trajectory in acute myeloid leukemia (AML) microenvironment is essential for understanding mechanisms of evading immune surveillance and immunotherapy response of targeting special microenvironment components. Methods Using a single-cell RNA sequencing (scRNA-seq) dataset, we analyzed the immune cell phenotypes, function, and developmental trajectory of bone marrow (BM) samples from 16 AML patients and 4 healthy donors, but not AML blasts. Results We observed a significant difference between normal and AML BM immune cells. Here, we defined the diversity of dendritic cells (DC) and macrophages in different AML patients. We also identified several unique immune cell types including T helper cell 17 (TH17)-like intermediate population, cytotoxic CD4+ T subset, T cell: erythrocyte complexes, activated regulatory T cells (Treg), and CD8+ memory-like subset. Emerging AML cells remodels the BM immune microenvironment powerfully, leads to immunosuppression by accumulating exhausted/dysfunctional immune effectors, expending immune-activated types, and promoting the formation of suppressive subsets. Conclusion Our results provide a comprehensive AML BM immune cell census, which can help to select pinpoint targeted drug and predict efficacy of immunotherapy.

Ginseng has beneficial effects on the cardiovascular system, but its underlying mechanism is unclear. This study investigated the effects of ginsenoside Rb1, a major constituent of ginseng, on the changes of lactate dehydrogenase (LDH) activity, nitric oxide (NO), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor-1 (PAI-1) in oxidized low-density lipoprotein (oxLDL)-injuring endothelial cells. Human umbilical vein endothelial cells were cultured and divided into 6 groups (n = 6): control group, oxLDL alone group (100 mg/L), ginsenoside Rb1 alone group (10 microg/mL), oxLDL plus ginsenoside Rb1 groups (0.1, 1.0, and 10 microg/mL, respectively.). Twenty-four hours after treatment, LDH activity and concentrations of NO, t-PA, and PAI-1 in culture medium were measured while the expressions of endothelial nitric oxide synthase (eNOS), t-PA, and PAI-1 mRNA in endothelial cells were detected by reverse-transcriptase polymerase chain reaction. Compared with control group, oxLDL (100 mg/L) caused LDH activity, the expressions of eNOS and t-PA mRNA, and concentrations of NO and t-PA to significantly decrease (P < 0.05, respectively), and it also led to dramatic increase of PAI-1 mRNA and concentration (P < 0.05, respectively). Ginsenoside Rb1 alone did not demonstrate this ability. High-dose Rb1 (10 microg/mL) could block the effects of oxLDL on LDH activity, mRNA of eNOS, t-PA, and PAI-1, and concentrations of NO, t-PA, and PAI-1 (P < 0.05, respectively), and neither low-dose Rb1 (0.1 microg/mL) nor medium-dose Rb1 (1.0 microg/mL) demonstrated this ability. We conclude that ginsenoside Rb1 has protective effects on oxLDL-injuring human vascular endothelial cells and can reverse the effects of oxLDL on NO, t-PA, and PAI-1.

Highlights The value of swab types on the detection of SARS-CoV-2 from patients during infection late stage is studied. The effect of specimen collection time on the detection rate of novel coronavirus was explored. Nasopharyngeal /nasal swabs collected before washing in the morning are more suitable for screening of large-scale specimens.

Vascular calcification (VC) is a pathological process underpinning major cardiovascular conditions and has attracted public attention due to its high morbidity and mortality. Chronic kidney disease (CKD) is a common disease related to VC. Ginsenoside Rb1 (Rb1) has been reported to protect the cardiovascular system against vascular diseases, yet its role in VC and the underlying mechanisms remain unclear. In this study, we established a CKD‐associated VC rat model and a β‐glycerophosphate (β‐GP)‐induced vascular smooth muscle cell (VSMC) calcification model to investigate the effects of Rb1 on VC. Our results demonstrated that Rb1 ameliorated calcium deposition and VSMC osteogenic transdifferentiation both in vivo and in vitro. Rb1 treatment inhibited the Wnt/β‐catenin pathway by activating peroxisome proliferator‐activated receptor‐γ (PPAR‐γ), and confocal microscopy was used to show that Rb1 inhibited β‐catenin nuclear translocation in VSMCs. Furthermore, SKL2001, an agonist of the Wnt/β‐catenin pathway, compromised the vascular protective effect of Rb1. GW9662, a PPAR‐γ antagonist, reversed Rb1's inhibitory effect on β‐catenin. These results indicate that Rb1 exerted anticalcific properties through PPAR‐γ/Wnt/β‐catenin axis, which provides new insights into the potential theraputics of VC.

Some driver gene mutations, including epidermal growth factor receptor ( EGFR ), have been reported to be involved in expression regulation of the immunosuppressive checkpoint protein programmed cell death ligand 1 ( PD ‐L1), but the underlying mechanism remains obscure. We investigated the potential role and precise mechanism of EGFR mutants in PD ‐L1 expression regulation in non‐small‐cell lung cancer ( NSCLC ) cells. Examination of pivotal EGFR signaling effectors in 8 NSCLC cell lines indicated apparent associations between PD ‐L1 overexpression and phosphorylation of AKT and ERK , especially with increased protein levels of phospho‐IκBα (p‐IκBα) and hypoxia‐inducible factor‐1α (HIF‐1α). Flow cytometry results showed stronger membrane co‐expression of EGFR and PD ‐L1 in NSCLC cells with EGFR mutants compared with cells carrying WT EGFR . Additionally, ectopic expression or depletion of EGFR mutants and treatment with EGFR pathway inhibitors targeting MEK / ERK , PI 3K/ AKT , mTOR /S6, IκBα, and HIF ‐1α indicated strong accordance among protein levels of PD ‐L1, p‐IκBα, and HIF ‐1α in NSCLC cells. Further treatment with pathway inhibitors significantly inhibited xenograft tumor growth and p‐IκBα, HIF ‐1α, and PD ‐L1 expression of NSCLC cells carrying EGFR mutant in nude mice. Moreover, immunohistochemical analysis revealed obviously increased protein levels of p‐IκBα, HIF ‐1α, and PD ‐L1 in NSCLC tissues with EGFR mutants compared with tissues carrying WT EGFR . Non‐small‐cell lung cancer tissues with either p‐IκBα or HIF ‐1α positive staining were more likely to possess elevated PD ‐L1 expression compared with tissues scored negative for both p‐IκBα and HIF ‐1α. Our findings showed important roles of phosphorylation activation of AKT and ERK and potential interplay and cooperation between NF ‐κB and HIF ‐1α in PD ‐L1 expression regulation by EGFR mutants in NSCLC .

This study was designed to explore whether exosomal sphingosine 1‐phosphate (S1P) from mesenchymal stem cells (MSCs) regulate the Treg/Th17 balance in aplastic anemia (AA) patients and to validate the underlying mechanism. To address this, exosomes from human bone marrow MSCs (MSCs‐Exos) were co‐cultured with CD4+ T cells from AA patients (AA CD4+ T cells), which were transfected with si‐S1PR1, si‐S1PR3, or not. The proportion of Th17 and Treg was evaluated by flow cytometry. The levels of Th17‐associated interleukin‐17 (IL‐17), Treg‐associated IL‐10, and transforming growth factor‐β were determined by ELISA. S1P content in MSCs‐Exos isolated from control, si‐SphK1, or si‐SphK2 transfected MSCs was examined by LC–MS/MS. Hematoxylin and eosin staining of bone marrow tissues was performed to evaluate the effect of MSCs‐Exos in AA mice. Our results showed that MSCs‐Exos reversed the increased Th17/Treg in AA through SphK1‐mediated exosomal S1P enrichment. Furthermore, the promotion of Treg differentiation by exosomal S1P from MSCs was mediated through the receptor S1PR1 expressed on CD4+ T cells. Further in vivo experiments showed that MSCs‐Exos reversed the increased Th17/Treg and alleviated AA progression in AA mice. In summary, SphK1‐mediated enrichment of exosomal S1P secreted by MSCs reversed the increased Treg/Th17 ratio via the receptor S1PR1 in AA patients. © 2019 IUBMB Life, 71(9):1284–1292, 2019

To investigate the U2AF1 gene mutation site, mutation load and co-mutations genes in patients with myelodysplastic syndrome (MDS) and their effects on prognosis. Gene mutation detection by next-generation sequence and related clinical data of 234 MDS patients were retrospectively collected and analyzed for the relationship between the clinical characteristics, treatment efficacy and prognosis of U2AF1 gene mutation. Among the 234 MDS patients, the U2AF1 gene mutation rate was 21.7% (51 cases), and the median variant allele frequency was 39.5%. Compared with the wild type, the U2AF1 mutant had a higher incidence of chromosome 8 aberration, and was positively correlated with the occurrence of ASXL1, RUNX1, SETBP1 gene mutation, negatively correlated with SF3B1, NPM1 genes mutation (p < 0.05). The most common mutation site of U2AF1 was S34F (32 cases), while U2AF1 Q157P site mutations had a higher incidence of chromosome 7 abnormalities (p = 0.003). The U2AF1 gene mutation more frequently coincided with signal pathway related gene mutations (p = 0.043) with a trend of shortened overall survival. Among patients with U2AF1 gene mutations, those with ASXL1 mutations were prone to develop into acute myeloid leukemia, those with RUNX1 mutations had an increased risk of relapse, and those with TET2 mutations had higher 1-year survival rate. Compared with the patient group of lower mutation load (VAF ≤ 40%), the group with higher mutation load of U2AF1 (VAF > 40%) had a significantly lower 1-year survival rate (46.1% and 80.5%, p = 0.027). The criteria of U2AF1 VAF > 40% is an independent indicator for poor prognosis of MDS patients. VAF > 40% of U2AF1 is an independent factor of short OS in MDS patients. MDS patients with a mutation in the Q157P site of U2AF1 and a higher U2AF1 mutation load suggests poor prognosis, and co-mutated genes in U2AF1 can affect disease progression and prognosis.