Cephalotaxine (CET) is a natural alkaloid with potent antileukemia effects. However, its underlying molecular mechanism has not been well understood. In this study, we verified that CET significantly inhibited the viability of various leukemia cells, including HL-60, NB4, Jurkat, K562, Raji and MOLT-4. RNA-sequencing and bioinformatics analysis revealed that CET causes mitochondrial function change. Mechanism research indicated that CET activated the mitochondrial apoptosis pathway by reducing the mitochondrial membrane potential, downregulating anti-apoptotic Bcl-2 protein and upregulating pro-apoptotic Bak protein. In addition, the autophagy signaling pathway was highly enriched by RNA-seq analysis. Then, we found that CET blocked the fluorescence colocation of MitoTracker Green and LysoTracker Red and upregulated the level of LC3-II and p62, which indicated that autophagy flow was impaired. Further results demonstrated that CET could impair lysosomal acidification and block autophagy flow. Finally, inhibiting autophagy flow could aggravate apoptosis of HL-60 cells induced by CET. In summary, this study demonstrated that CET exerted antileukemia effects through activation of the mitochondria-dependent pathway and by impairing autophagy flow. Our research provides new insights into the molecular mechanisms of CET in the treatment of leukemia.

This study aimed to improve the transdermal delivery of lidocaine hydrochloride (LidH) using elastic nano-liposomes (ENLs) and microneedle (MN) array pretreatment. LidH-containing ENLs were prepared using soybean phosphatidylcholine and cholesterol, with Span 80 or Tween 80, using a reverse-phase evaporation method. The ENL particle size, stability, and encapsulation efficiency (EE) were characterized and optimized based on the component ratio, pH, and type of surfactant used. In vitro transdermal diffusion study was performed on MN-pretreated mouse skin using Franz diffusion cells. The anesthetic effects of LidH in various formulations after dermal application were evaluated in vivo in rats by measuring the tail withdrawal latency after photothermic stimulation. Stable LidH-loaded Tween 80 or Span 80 ENLs were obtained with particle sizes of 115.8 and 146.6 nm and EEs of 27% and 20%, respectively. The formulations did not exert any cytotoxicity in HaCaT cells. Tween 80 and Span 80 ENL formulations showed enhanced LidH delivery on pretreated mice skin in vitro and prolonged the anesthetic effect in vivo compared to that by LidH application alone. LidH-loaded ENLs applied to MN-pretreated skin can shorten the onset time and prolong the anesthetic effect safely, which merits their further optimization and practical application.

Prognosis of patients for human epidermal growth factor receptor 2 (HER2)-negative breast cancer post neoadjuvant chemotherapy is not well understood. The aim of this study was to develop a novel pharmacophore-based signature to better classify and predict the risk of HER2-negative patients after anthracycline-and/or taxane-based neoadjuvant chemotherapy (NACT).

Eukaryotic cells contain numerous membraneless organelles that are made from liquid droplets of proteins and nucleic acids and that provide spatiotemporal control of various cellular processes. However, the molecular mechanisms underlying the formation and rapid stress-induced alterations of these organelles are relatively uncharacterized. Here, we investigated the roles of DEAD-box helicases in the formation and alteration of membraneless nuclear dicing bodies (D-bodies) in Arabidopsis thaliana We uncovered that RNA helicase 6 (RH6), RH8, and RH12 are previously unidentified D-body components. These helicases interact with and promote the phase separation of SERRATE, a key component of D-bodies, and drive the formation of D-bodies through liquid-liquid phase separations (LLPSs). The accumulation of these helicases in the nuclei decreases upon Turnip mosaic virus infections, which couples with the decrease of D-bodies. Our results thus reveal the key roles of RH6, RH8, and RH12 in modulating D-body formation via LLPSs.

Cervical cancer is the fourth most prevalent cancer in women, which decreases quality of life of the patients. Traditional interventions have failed to improve the overall survival period of patients due to high tumor recurrence after treatment or late diagnosis. Fortunately, preliminary evidence suggests that anti-angiogenic and immunotherapy can efficiently treat against cervical cancer. However, there is no clear evidence on the efficacy of immunotherapy in cervical cancer. Therefore, in this study, we classified cervical cancers in the TCGA dataset using various algorithms and explored the relationship between the immune profile and corresponding sensitivity of the tumors to immunotherapy. Results showed that patients with tumors had higher expression of immunocytes and longer overall survival time. In addition, we build a scoring system based on the immune landscape of the tumor microenvironment of cervical cancer. Tumors with higher scores exhibited better survival outcomes and were more sensitive to immunotherapy. In this study, the immune landscape of cervical cancer was analyzed, and the subtype of cervical cancer based on that difference was proposed. Besides, the subtype of cervical cancer showed different sensitivity to immunotherapeutic response which further confirmed its relationship with tumor immune landscape.

Loss of perivascular adipose tissue (PVAT) impairs endothelial function and enhances atherosclerosis. However, the roles of PVAT thermoregulation in vascular inflammation and the development of atherosclerosis remains unclear. Bone morphogenetic protein 4 (BMP4) transforms white adipocyte to beige adipocyte, while promotes a brown-to-white shift in inter-scapular brown adipose tissue (BAT). Here, we found that knockdown of BMP4 in PVAT reduced expression of brown adipocyte-characteristic genes and increased endothelial inflammation in vitro co-culture system. Ablating BMP4 expression either in adipose tissues or specifically in BAT in ApoE-/- mice demonstrated a marked exacerbation of atherosclerotic plaque formation in vivo. We further demonstrated that proinflammatory factors (especially IL-1β) increased in the supernatant of BMP4 knockdown adipocytes. Overexpression of BMP4 in adipose tissues promotes browning of PVAT and protects against atherosclerosis in ApoE-/- mice. These findings uncover an organ crosstalk between PVAT and blood endothelial cells that is engaged in atherosclerosis.

Based on 1572 re-sequenced Chinese tongue sole (Cynoglossus semilaevis), we investigated the accuracy of four genomic methods at predicting genomic estimated breeding values (GEBVs) of Vibrio harveyi resistance in C. semilaevis when SNPs varying from 500 to 500 k. All methods outperformed the pedigree-based best linear unbiased prediction when SNPs reached 50 k or more. Then, we developed an SNP array "Solechip No.1" for C. semilaevis breeding using the Affymetrix Axiom technology. This array contains 38,295 SNPs with an average of 10.5 kb inter-spacing between two adjacent SNPs. We selected 44 candidates as the parents of 23 families and genotyped them by the array. The challenge survival rates of offspring families had a correlation of 0.706 with the mid-parental GEBVs. This SNP array is a convenient and reliable tool in genotyping, which could be used for improving V. harveyi resistance in C. semilaevis coupled with the genomic selection methods.

The objective of the study was to detect the expression of Kita-Kyushu lung cancer antigen-1 (KK-LC-1) in gastric cancer (GC) specimens and analyse the associations between KK-LC-1 expression and clinicopathological parameters and clinical prognosis.

T cells play a critical role in coronavirus diseases. How they do so in COVID-19 may be revealed by analyzing the epigenetic chromatin accessibility of cis- and trans-regulatory elements and creating transcriptomic immune profiles. We performed single-cell assay for transposase-accessible chromatin (scATAC) and single-cell RNA (scRNA) sequencing (seq) on the peripheral blood mononuclear cells (PBMCs) of severely ill/critical patients (SCPs) infected with COVID-19, moderate patients (MPs), and healthy volunteer controls (HCs). About 76,570 and 107,862 single cells were used, respectively, for analyzing the characteristics of chromatin accessibility and transcriptomic immune profiles by the application of scATAC-seq (nine cases) and scRNA-seq (15 cases). The scATAC-seq detected 28,535 different peaks in the three groups; among these peaks, 41.6 and 10.7% were located in the promoter and enhancer regions, respectively. Compared to HCs, among the peak-located genes in the total T cells and its subsets, CD4+ T and CD8+ T cells, from SCPs and MPs were enriched with inflammatory pathways, such as mitogen-activated protein kinase (MAPK) signaling pathway and tumor necrosis factor (TNF) signaling pathway. The motifs of TBX21 were less accessible in the CD4+ T cells of SCPs compared with those in MPs. Furthermore, the scRNA-seq showed that the proportion of T cells, especially the CD4+ T cells, was decreased in SCPs and MPs compared with those in HCs. Transcriptomic results revealed that histone-related genes, and inflammatory genes, such as NFKBIA, S100A9, and PIK3R1, were highly expressed in the total T cells, CD4+ T and CD8+ T cells, both in the cases of SCPs and MPs. In the CD4+ T cells, decreased T helper-1 (Th1) cells were observed in SCPs and MPs. In the CD8+T cells, activation markers, such as CD69 and HLA class II genes (HLA-DRA, HLA-DRB1, and HLA-DRB5), were significantly upregulated in SCPs. An integrated analysis of the data from scATAC-seq and scRNA-seq showed some consistency between the approaches. Cumulatively, we have generated a landscape of chromatin epigenetic status and transcriptomic immune profiles of T cells in patients with COVID-19. This has provided a deeper dissection of the characteristics of the T cells involved at a higher resolution than from previously obtained data merely by the scRNA-seq analysis. Our data led us to suggest that the T-cell inflammatory states accompanied with defective functions in the CD4+ T cells of SCPs may be the key factors for determining the pathogenesis of and recovery from COVID-19.

Neuroinflammation has been recognized as a major cause for neurocognitive diseases. Although the hippocampus has been considered an important region for cognitive dysfunction, the influence of hippocampal neuroinflammation on brain functional connectivity (FC) has been rarely studied. In this study, lipopolysaccharide (LPS) was used to induce systemic inflammation and neuroinflammation in the aged rat brain, while elamipretide (SS-31) was used for treatment. Systemic and hippocampal inflammation were determined using ELISA, while astrocyte responses during hippocampal neuroinflammation were determined by interleukin 1 beta (IL-1β)/tumor necrosis factor alpha (TNFα) double staining immunofluorescence. Oxidative stress was determined by reactive oxidative species (ROS), electron transport chain (ETC) complex, and superoxide dismutase (SOD). Short- (<7 days) and long-term (>30 days) learning and spatial working memory were tested by the Morris water maze (MWM). Resting-state functional magnetic resonance imaging (rs-fMRI) was used to analyze the brain FC by placing seed voxels on the left and right hippocampus. Compared with the vehicle group, rats with the LPS exposure showed an impaired MWM performance, higher oxidative stress, higher levels of inflammatory cytokines, and astrocyte activation in the hippocampus. The neuroimaging examination showed decreased FC on the right orbital cortex, right olfactory bulb, and left hippocampus on day 3, 7, and 31, respectively, after treatment. In contrast, rats with SS-31 treatment showed lower levels of inflammatory cytokines, less astrocyte activation in the hippocampus, and improved MWM performance. Neuroimaging examination showed increased FC on the left-parietal association cortex (L-PAC), left sensory cortex, and left motor cortex on day 7 with the right flocculonodular lobe on day 31 as compared with those without SS-31 treatment. Our study demonstrated that inhibiting neuroinflammation in the hippocampus not only reduces inflammatory responses in the hippocampus but also improves the brain FC in regions related to the hippocampus. Furthermore, early anti-inflammatory treatment with SS-31 has a long-lasting effect on reducing the impact of LPS-induced neuroinflammation.

Pseudomonas aeruginosa is a biofilm-forming opportunistic pathogen which causes chronic infections in immunocompromised patients and leads to high mortality rate. It is identified as a common coinfecting pathogen in COVID-19 patients causing exacerbation of illness. In our hospital, P. aeruginosa is one of the top coinfecting bacteria identified among COVID-19 patients. We collected a strong biofilm-forming P. aeruginosa strain displaying small colony variant morphology from a severe COVID-19 patient. Genomic and transcriptomic sequencing analyses were performed with phenotypic validation to investigate its adaptation in SARS-CoV-2 infected environment. Genomic characterization predicted specific genomic islands highly associated with virulence, transcriptional regulation, and DNA restriction-modification systems. Epigenetic analysis revealed a specific N6-methyl adenine (m6A) methylating pattern including methylation of alginate, flagellar and quorum sensing associated genes. Differential gene expression analysis indicated that this isolate formed excessive biofilm by reducing flagellar formation (7.4 to 1,624.1 folds) and overproducing extracellular matrix components including CdrA (4.4 folds), alginate (5.2 to 29.1 folds) and Pel (4.8-5.5 folds). In summary, we demonstrated that P. aeuginosa clinical isolates with novel epigenetic markers could form excessive biofilm, which might enhance its antibiotic resistance and in vivo colonization in COVID-19 patients.

Currently, there are no approved drugs for the treatment of flavivirus infection. Accordingly, we tested the inhibitory effects of the novel θ-defensin retrocyclin-101 (RC-101) against flavivirus infection and investigated the mechanism underlying the potential inhibitory effects. First, RC-101 robustly inhibited both Japanese encephalitis virus (JEV) and Zika virus (ZIKV) infections. RC-101 exerted inhibitory effects on the entry and replication stages. Results also indicated that the nonstructural protein NS2B-NS3 serine protease might serve as a potential viral target. Furthermore, RC-101 inhibited protease activity at the micromolar level. We also demonstrated that with respect to the glycoprotein E protein of flavivirus, the DE loop of domain III (DIII), which is the receptor-binding domain of the E protein, might serve as another viral target of RC-101. Moreover, a JEV DE mutant exhibited resistance to RC-101, which was associated with deceased binding affinity of RC-101 to DIII. These findings provide a basis for the development of RC-101 as a potential candidate for the treatment of flavivirus infection. IMPORTANCE Retrocyclin is an artificially humanized circular θ-defensin peptide, containing 18 residues, previously reported to possess broad antimicrobial activity. In this study, we found that retrocyclin-101 inhibited flavivirus (ZIKV and JEV) infections. Retrocyclin-101 inhibited NS2B-NS3 serine protease activity, suggesting that the catalytic triad of the protease is the target. Moreover, retrocyclin-101 bound to the DE loop of the E protein of flavivirus, which prevented its entry.

Hepatitis B virus (HBV) infection is a worldwide health problem with high morbidity and mortality rates. The therapeutic vaccine is a promising method of treatment, and HBV polymerase plays a vital role in viral replication. Therefore, a therapeutic vaccine that binds to HBV DNA polymerase may control HBV infection. We predicted and selected epitopes of polymerase using online databases and analysis software. We then performed molecular docking and peptide binding assays to evaluate the binding energies and affinities between polymerase epitopes and the HLA-A0201 molecule. Finally, we induced T cells from the peripheral blood mononuclear cells (PBMCs) of healthy donors using each epitope and quantified the functions of epitope-specific T cells by IFN-γELISPOT assay, T2 cell cytotoxicity assay, HepG2.2.15 cell cytotoxicity assay and HBV gene expression assays. Four epitopes (RVTGGVFLV, GLLGFAAPF, LLDDEAGPL and YMDDVVLGA) had low binding energy and two epitopes (RVTGGVFLV and GLLGFAAPF) had a high binding affinity. The T cells stimulated by two epitopes (GLLGFAAPF and HLYSHPIIL) had a greater ability to induce immune response and suppress HBV. The HBV DNA polymerase epitopes identified in this study are promising targets for designing an epitope-based therapeutic vaccine against HBV.

Our previous clinical study showed that low-dose decitabine exhibited sustained responses in nearly half of patients with refractory immune thrombocytopenia (ITP). The long-term efficacy of decitabine in ITP is not likely due to its simple role in increasing platelet production. Whether decitabine has the potential to restore immune tolerance in ITP is unknown. In this study, we analyzed the effect of decitabine on T-cell subpopulations in ITP in vitro and in vivo. We found that low-dose decitabine promoted the generation and differentiation of regulatory T (Treg) cells and augmented their immunosuppressive function. Splenocytes from CD61 knockout mice immunized with CD61+ platelets were transferred into severe combined immunodeficient mouse recipients to induce a murine model of ITP. Low-dose decitabine alleviated thrombocytopenia and restored the balance between Treg and helper T (Th) cells in active ITP mice. Treg deletion and depletion offset the effect of decitabine in restoring CD4+ T-cell subpopulations in ITP mice. For patients who received low-dose decitabine, the quantity and function of Treg cells were substantially improved, whereas Th1 and Th17 cells were suppressed compared with the pretreatment levels. Next-generation RNA-sequencing and cytokine analysis showed that low-dose decitabine rebalanced T-cell homeostasis, decreased proinflammatory cytokines, and downregulated phosphorylated STAT3 in patients with ITP. STAT3 inhibition analysis suggested that low-dose decitabine might restore Treg cells by inhibiting STAT3 activation. In conclusion, our data indicate that the immunomodulatory effect of decitabine provides one possible mechanistic explanation for the sustained response achieved by low-dose decitabine in ITP.

The potential treatment effects and safety of Yu ping feng san (YPFS) for pediatric allergic rhinitis (PAR) patients have yet to be studied systematically.

Annexin A4 (ANXA4) is a Ca2+- and phospholipid-binding protein that belongs to the annexin family, which is involved in the development of multiple tumour types via NF-κB signalling. In this study, we verified the high expression and membrane-cytoplasm translocation of ANXA4 in colorectal carcinoma (CRC). Calcium/calmodulin-dependent protein kinase II gamma (CAMK2γ) was found to be important for high ANXA4 expression in CRC, whereas carbonic anhydrase (CA1) promoted ANXA4 aggregation in the cell membrane. An increased Ca2+ concentration attenuated the small ubiquitin-like modifier (SUMO) modification of cytoplasmic ANXA4 and ANXA4 stabilization, and relatively high expression of ANXA4 promoted CRC tumorigenesis and epithelial-mesenchymal transition (EMT).

High rate of cardiovascular disease (CVD) has been reported among patients with coronavirus disease 2019 (COVID-19). Importantly, CVD, as one of the comorbidities, could also increase the risks of the severity of COVID-19. Here we identified phospholipase A2 group VII (PLA2G7), a well-studied CVD biomarker, as a hub gene in COVID-19 though an integrated hypothesis-free genomic analysis on nasal swabs (n = 486) from patients with COVID-19. PLA2G7 was further found to be predominantly expressed by proinflammatory macrophages in lungs emerging with progression of COVID-19. In the validation stage, RNA level of PLA2G7 was identified in nasal swabs from both COVID-19 and pneumonia patients, other than health individuals. The positive rate of PLA2G7 were correlated with not only viral loads but also severity of pneumonia in non-COVID-19 patients. Serum protein levels of PLA2G7 were found to be elevated and beyond the normal limit in COVID-19 patients, especially among those re-positive patients. We identified and validated PLA2G7, a biomarker for CVD, was abnormally enhanced in COVID-19 at both nucleotide and protein aspects. These findings provided indications into the prevalence of cardiovascular involvements seen in patients with COVID-19. PLA2G7 could be a potential prognostic and therapeutic target in COVID-19.

Beginning in the summer of 2020, a variant of SARS-CoV-2, the cause of the COVID-19 pandemic, emerged in the United Kingdom (UK). This B.1.1.7 variant increased rapidly in prevalence among sequenced strains, attributed to an increase in infection and/or transmission efficiency. The UK variant has 19 nonsynonymous mutations across its viral genome including 8 substitutions or deletions in the spike protein, which interacts with cellular receptors to mediate infection and tropism. Here, using a reverse genetics approach, we show that, of the 8 individual spike protein substitutions, only N501Y exhibited consistent fitness gains for replication in the upper airway in the hamster model as well as primary human airway epithelial cells. The N501Y substitution recapitulated the phenotype of enhanced viral transmission seen with the combined 8 UK spike mutations, suggesting it is a major determinant responsible for increased transmission of this variant. Mechanistically, the N501Y substitution improved the affinity of the viral spike protein for cellular receptors. As suggested by its convergent evolution in Brazil and South Africa, our results indicate that N501Y substitution is a major adaptive spike mutation of major concern.

Oral lichen planus (OLP), defined as a potential for malignant transformation, is a chronic inflammatory disease in which abnormal angiogenesis serves a role in the malignant changes of the disease. OLP-associated fibroblasts (OLP-MFs), derived from the stroma of OLP tissues, are characterized by the presence of myofibroblasts and contribute to the secretion of pro-inflammatory cytokines, which may be involved in the molecular pathogenesis of OLP. However, the associated mechanisms of angiogenesis in OLP remain unknown. The present study aimed to verify the expression of intercellular adhesion molecular 1, vascular cell adhesion molecule 1, VEGF and CD34 in OLP, and to investigate whether IL-6 secreted by OLP-MFs promoted OLP angiogenesis and the effect of its corresponding antibody inhibition. The results of the experiments demonstrated that inflammation was present and OLP upregulated the secretion of IL-6 by OLP stromal fibroblasts, thereby enhancing OLP angiogenesis. Anti-IL-6 receptor antibody inhibited OLP-stroma IL-6 signaling and suppressed OLP angiogenesis. The antibody inhibited the inflammatory response by inhibiting the secretion of inflammatory factors, including IL-6, to suppress angiogenesis and reduce disease progression, thus indicating that this could be a potential target to develop a treatment for OLP.

Soluble sugars and starches are important metabolites of plant life and physiological markers of plant stress response. There is an urgent need to develop a non-destructive and rapid method for determining plant starch and soluble sugar contents. Electrical impedance tomography (EIT) technology has been used to determine the physiological state and cold resistance of select plant tissues. However, so far there have been no reports on the use of EIT for the rapid estimation of soluble sugar and starch contents. In this study, EIT was used to obtain reconstructed voltage values and estimate starch and soluble sugar contents in the stems of three Rosa hybrida cultivars during February to May, which were grown in the Specimen Park (38° 50' N, 115° 26' E) of Hebei Agricultural University, Baoding City, Hebei Province, China. Stems from two of the cultivars were used for establishing regression models for starch and soluble sugar contents as functions of reconstructed voltage values. The third cultivar was used to test the accuracy of the regression models. The quadratic regression model was best for determining soluble sugar content and the logarithmic regression model was best for determining starch content. Thus, this research provided technical support for using EIT to analyze changes in physiological parameters and to rapidly estimate physiological indexes of plants. More studies were now needed to validate the results in this paper.