METTL3-mediated m6A methylation regulates ovarian cancer progression by recruiting myeloid-derived suppressor cells.

BACKGROUND: Ovarian cancer (OC) typically develops an immunosuppressive microenvironment by funtional changes of host immune cells. Dysregulated m6A level is associated with cancer progression via the intrinsic oncogenic pathways. However, the role of m6A in regulating host immune cell function during anti-tumor immunity needs comprehensive analysis. This study aimed to investigate the role of METTL3, a catalytic subunit of the methyltransferase complex, in regulating host immune cell response against OC. METHODS: In this study, myeloid-specific Mettl3 gene knockout (Mettl3-cKO) mice were bred using the Cre-LoxP system. Intraperitoneally injection of ID8 cells was used as a syngeneic OC model. Furthermore, the compositions of immune cell populations were analyzed by flow cytometry and single-cell sequencing. Moreover, chemokines and cytokines secretion were assessed using ELISA. Lastly, the role of METTL3 in regulating IL-1ß secretion and inflammasome activation in bone marrow-derived macrophages cocultured with ID8 cells was specified by ELISA and immunoblotting. RESULTS: It was revealed that OC cell growth was enhanced in Mettl3-cKO mice. Furthermore, a shift of decreased M1 to increased M2 macrophage polarization was observed during OC progression. Moreover, Mettl3 depletion in myeloid lineage cells increased secretion of CCL2 and CXCL2 in peritoneal lavage fluild. Interestingly, Mettl3 deficiency enhanced IL-1ß secretion induced by viable ID8 cells independent of inflammasome activation and cell death. Therefore, OC cells in tumor-bearing mice trigger a slight inflammatory response with a low-to-moderate secretion of pro-inflammatory cytokines and chemokines. CONCLUSION: This study provides new insights into METTL3-mediated m6A methylation, which regulates host immune response against OC.

Accurate classification of pulmonary nodules by a combined model of clinical, imaging, and cell-free DNA methylation biomarkers: a model development and external validation study.

BACKGROUND: There is an unmet clinical need for accurate non-invasive tests to facilitate the early diagnosis of lung cancer. We propose a combined model of clinical, imaging, and cell-free DNA methylation biomarkers that aims to improve the classification of pulmonary nodules. METHODS: We conducted a prospective specimen collection and retrospective masked evaluation study. We recruited participants with a solitary pulmonary nodule sized 5-30 mm from 24 hospitals across 20 cities in China. Participants who were aged 18 years or older and had been referred with 5-30 mm non-calcified and solitary pulmonary nodules, including solid nodules, part solid nodules, and pure ground-glass nodules, were included. We developed a combined clinical and imaging biomarkers (CIBM) model by machine learning for the classification of malignant and benign pulmonary nodules in a cohort (n=839) and validated it in two cohorts (n=258 in the first cohort and n=283 in the second cohort). We then integrated the CIBM model with our previously established circulating tumour DNA methylation model (PulmoSeek) to create a new combined model, PulmoSeek Plus (n=258), and verified it in an independent cohort (n=283). The clinical utility of the models was evaluated using decision curve analysis. A low cutoff (0·65) for high sensitivity and a high cutoff (0·89) for high specificity were applied simultaneously to stratify pulmonary nodules into low-risk, medium-risk, and high-risk groups. The primary outcome was the diagnostic performance of the CIBM, PulmoSeek, and PulmoSeek Plus models. Participants in this study were drawn from two prospective clinical studies that were registered (NCT03181490 and NCT03651986), the first of which was completed, and the second of which is ongoing because 25% of participants have not yet finished the required 3-year follow-up. FINDINGS: We recruited a total of 1380 participants. 1097 participants were enrolled from July 7, 2017, to Feb 12, 2019; 839 participants were used for the CIBM model training set, and the rest (n=258) for the first CIBM validation set and the PulmoSeek Plus training set. 283 participants were enrolled from Oct 26, 2018, to March 20, 2020, as an independent validation set for the PulmoSeek Plus model and the second validation set for the CIBM model. The CIBM model validation cohorts had area under the curves (AUCs) of 0·85 (95% CI 0·80-0·89) and 0·85 (0·81-0·89). The PulmoSeek Plus model had better discrimination capacity compared with the CIBM and PulmoSeek models with an increase of 0·05 in AUC (PulmoSeek Plus vs CIBM, 95% CI 0·022-0·087, p=0·001; and PulmoSeek Plus vs PulmoSeek, 0·018-0·083, p=0·002). The overall sensitivity of the PulmoSeek Plus model was 0·98 (0·97-0·99) at a fixed specificity of 0·50 for ruling out lung cancer. A high sensitivity of 0·98 (0·96-0·99) was maintained in early-stage lung cancer (stages 0 and I) and 0·99 (0·96-1·00) in 5-10 mm nodules. The decision curve showed that if an invasive intervention, such as surgical resection or biopsy, was deemed necessary at more than the risk threshold score of 0·54, the PulmoSeek Plus model would provide a standardised net benefit of 82·38% (76·06-86·79%), equivalent to correctly identifying approximately 83 of 100 people with lung cancer. Using the PulmoSeek Plus model to classify pulmonary nodules with two cutoffs (0·65 and 0·89) would have reduced 89% (105/118) of unnecessary surgeries and 73% (308/423) of delayed treatments. INTERPRETATION: The PulmoSeek Plus Model combining clinical, imaging, and cell-free DNA methylation biomarkers aids the early diagnosis of pulmonary nodules, with potential application in clinical decision making for the management of pulmonary nodules. FUNDING: The China National Science Foundation, the Key Project of Guangzhou Scientific Research Project, the High-Level University Construction Project of Guangzhou Medical University, the National Key Research & Development Programme, the Guangdong High Level Hospital Construction "Reaching Peak" Plan, the Guangdong Basic and Applied Basic Research Foundation, the National Natural Science Foundation of China, The Leading Projects of Guangzhou Municipal Health Sciences Foundation, the Key Research and Development Plan of Shaanxi Province of China, the Scheme of Guangzhou Economic and Technological Development District for Leading Talents in Innovation and Entrepreneurship, the Scheme of Guangzhou for Leading Talents in Innovation and Entrepreneurship, the Scheme of Guangzhou for Leading Team in Innovation, the Guangzhou Development Zone International Science and Technology Cooperation Project, and the Science and Technology Planning Project of Guangzhou.

Accurate detection of early-stage lung cancer using a panel of circulating cell-free DNA methylation biomarkers.

BACKGROUND: Lung cancer remains the leading cause of cancer mortality worldwide. Early detection of lung cancer helps improve treatment and survival. Numerous aberrant DNA methylations have been reported in early-stage lung cancer. Here, we sought to identify novel DNA methylation biomarkers that could potentially be used for noninvasive early diagnosis of lung cancers. METHODS: This prospective-specimen collection and retrospective-blinded-evaluation trial enrolled a total of 317 participants (198 tissues and 119 plasmas) comprising healthy controls, patients with lung cancer and benign disease between January 2020 and December 2021. Tissue and plasma samples were subjected to targeted bisulfite sequencing with a lung cancer specific panel targeting 9,307 differential methylation regions (DMRs). DMRs associated with lung cancer were identified by comparing the methylation profiles of tissue samples from patients with lung cancer and benign disease. Markers were selected with minimum redundancy and maximum relevance algorithm. A prediction model for lung cancer diagnosis was built through logistic regression algorithm and validated independently in tissue samples. Furthermore, the performance of this developed model was evaluated in a set of plasma cell-free DNA (cfDNA) samples. RESULTS: We identified 7 DMRs corresponding to 7 differentially methylated genes (DMGs) including HOXB4, HOXA7, HOXD8, ITGA4, ZNF808, PTGER4, and B3GNTL1 that were highly associated with lung cancer by comparing the methylation profiles of lung cancer and benign nodule tissue. Based on the 7-DMR biomarker panel, we developed a new diagnostic model in tissue samples, termed "7-DMR model", to distinguish lung cancers from benign diseases, achieving AUCs of 0.97 (95%CI: 0.93-1.00)/0.96 (0.92-1.00), sensitivities of 0.89 (0.82-0.95)/0.92 (0.86-0.98), specificities of 0.94 (0.89-0.99)/1.00 (1.00-1.00), and accuracies of 0.90 (0.84-0.96)/0.94 (0.89-0.99) in the discovery cohort (n = 96) and the independent validation cohort (n = 81), respectively. Furthermore, the 7-DMR model was applied to noninvasive discrimination of lung cancers and non-lung cancers including benign lung diseases and healthy controls in an independent validation cohort of plasma samples (n = 106), yielding an AUC of 0.94 (0.86-1.00), sensitivity of 0.81 (0.73-0.88), specificity of 0.98 (0.95-1.00), and accuracy of 0.93 (0.89-0.98). CONCLUSION: The 7 novel DMRs could be promising methylation biomarkers that merits further development as a noninvasive test for early detection of lung cancer.

Black phosphorus-based photothermal therapy with aCD47-mediated immune checkpoint blockade for enhanced cancer immunotherapy.

Here, we describe a combination strategy of black phosphorus (BP)-based photothermal therapy together with anti-CD47 antibody (aCD47)-based immunotherapy to synergistically enhance cancer treatment. Tumour resistance to immune checkpoint blockades in most cancers due to immune escape from host surveillance, along with the initiation of metastasis through immunosuppressive cells in the tumour microenvironment, remains a significant challenge for cancer immunotherapy. aCD47, an agent for CD47/SIRPα axis blockade, induces modest phagocytic activity and a low response rate for monotherapy, resulting in failures in clinical trials. We showed that BP-mediated ablation of tumours through photothermal effects could serve as an effective strategy for specific immunological stimulation, improving the inherently poor immunogenicity of tumours, which is particularly useful for enhancing cancer immunotherapy. BP in combination with aCD47 blockade activates both innate and adaptive immunities and promotes local and systemic anticancer immune responses, thus offering a synergistically enhanced effect in suppression of tumour progression and in inducing abscopal effects for inhibition of metastatic cancers. Our combination strategy provides a promising platform in which photothermal agents could help to enhance the therapeutic efficacy of immunotherapy.

lncRNA HEIH accelerates cell proliferation and inhibits cell senescence by targeting miR-3619-5p/CTTNBP2 axis in ovarian cancer.

OBJECTIVE: Epithelial ovarian cancer is the most lethal malignancy in gynecology. Numerous studies have confirmed that long noncoding RNAs (lncRNAs) are abnormally expressed in ovarian cancer and are closely associated with the cell proliferation and senescence in cancers. However, the role and underlying molecular mechanism of long noncoding RNA high expression in hepatocellular carcinoma (HEIH) in ovarian cancer remain unknown. METHODS: Experiments including Real-time quantitative polymerase chain reaction, RNA immunoprecipitation, luciferase reporter, Fluorescence in situ hybridization, western blot, colony formation assays, ß-galactosidase senescence assay, cell apoptosis, proliferation, invasion, and migration assays were applied to investigate the role of HEIH in ovarian cancer. The data were expressed as the mean ±â€Šstandard deviation. Student t test was used to compare the data between two groups. The one-way analysis of variance was applied to compare the data among multiple groups with Tukey post hoc test. All experiments were repeated three times. P < 0.05 was considered statistically significant. RESULTS: Herein, HEIH expression was found to be up-regulated in ovarian cancer tissues (n = 25; twofold higher than normal tissues, P < 0.05) and cell lines (sixfold higher than normal ovarian epithelial cell line on average, P < 0.05), and high HEIH expression predicted poor prognosis (survival rate is about 25% after 40 mo; P < 0.05). Moreover, we found that HEIH accelerated proliferation, migration, and invasion, whereas inhibited cell senescence in ovarian cancer (P < 0.05). In mechanism, HEIH was confirmed to serve as a sponge for miR-3619-5p, and miR-3619-5p counteracted HEIH-mediated regulation of ovarian cancer (P < 0.05). Besides, cortactin-binding protein 2 (CTTNBP2) was found to be the downstream target of miR-3619-5p. Rescue assays validated that CTTNBP2 up-regulation significantly reversed the inhibitory effects of HEIH knockdown on ovarian cancer progression (P < 0.05). Furthermore, we found that HEIH facilitated tumor growth in vivo by regulating CTTNBP2 expression (P < 0.05). CONCLUSIONS: In conclusion, our research revealed that HEIH accelerated cell proliferation, migration and invasion, whereas inhibited cell senescence in ovarian cancer via targeting the miR-3619-5p/CTTNBP2 axis. These findings may be valuable for finding new therapeutic targets to improve ovarian cancer treatment.

Liquid-phase exfoliation of black sesame to create a nanoplatform for in vitro photoluminescence and photothermal therapy.

Aim: The present study aims to apply the facile liquid-phase exfoliation (LPE) strategy to fabricate 2D organic materials and thus to broaden the family of biocompatible and multifunctional 2D materials. Materials & methods: 2D material-organic melanin and cellulose nanosheets were synthesized from black sesame hull using LPE. Photoluminescence and photothermal properties of the nanosheets were assessed, as well as stability and cell killing ability. Results: The prepared 2D nanoplatform exhibited broad and multiple photoluminescent emission bands. It also demonstrated efficient photothermal cancer therapy with excellent biocompatibility. Conclusion: The present study could open an avenue in exfoliating organic materials using the LPE strategy. This could make the fabrication of multifunctional 2D organic materials more efficient and broaden the family of biocompatible 2D nanomaterials.

Outbreak of COVID-19: An emerging global pandemic threat.

Since the first outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Wuhan, Hubei, China in December 2019, it is now recognized as a pandemic by the World Health Organization (WHO) as more than 200 countries and territories worldwide are affected with an increasing incidence. The SARS-CoV-2 infection results in a spectrum of non-specific signs and symptoms, ranging from asymptomatic infection, to flu-like illness such as fever, cough, dry cough and fatigue, to pneumonia, acute respiratory distress syndrome, and even multi-organ failures with high morbidity and mortality. SARS-CoV-2 is mainly transmitted through respiratory droplets that infected people exhale during incubation and onset period. By 12 June 2020, over 7.5 million confirmed cases of Coronavirus disease 2019 (COVID-19) with more than 421,000 deaths in the world have been reported to the WHO. No specific medication is approved to treat COVID-19, raising the urgent need for antiviral drug development. By 12 June 2020, there are over 1000 clinical trials registered in clinicaltrials.gov for treatment of COVID-19. This review summarizes the epidemiology, virology, clinical presentation, pathophysiology, diagnosis, and particularly the antiviral drugs currently under clinical trials for treatment of SARS-CoV-2 infection, together with the challenges and perspectives of this disease are also discussed.

Luteolin escape mutants of dengue virus map to prM and NS2B and reveal viral plasticity during maturation.

We previously showed that luteolin, a well-known plant-derived component found in the "heat clearing" class of Traditional Chinese Medicine (TCM) herbs, is an uncompetitive inhibitor (Ki 58.6 µM) of the host proprotein convertase furin, an endoprotease that is required for maturation of flaviviruses in the trans-Golgi compartment. Luteolin also weakly inhibited recombinant dengue virus NS2B/NS3 protease (Ki 140.36 µM) non-competitively. In order to further explore the mechanism of inhibition we isolated resistant mutants by continuous passaging of DENV2 in the presence of increasing concentrations of luteolin. Nucleotide sequence analysis of the luteolin-resistant escape mutants revealed nucleotide changes that lead to amino acid substitutions in the prM (T79R) and NS2B (I114M) genes. These mutations were introduced into a DENV2 infectious clone and tested for replication in Huh-7 cells. Interestingly we found that the replication kinetics of prM T19R-NS2B I114M double-mutant (DM) was similar to wild-type virus (WT). On the other hand the prM T79R single mutant (SM1) was attenuated and the NS2B I114M single mutant (SM2) showed enhanced replication. Time of drug addition assay with luteolin showed that the mutant viruses were able to produce more mature virions than WT in the order DM > SM2>SM1>WT. Exogenous addition of furin to purified immature WT or mutant viruses revealed that luteolin blocked the prM cleavage of WT and SM2 at a similar level. On the other hand the SM1 immature virus showed some cleavage while the DM immature virus revealed efficient furin cleavage of prM even in the presence of 50 µM luteolin. Our findings suggest that luteolin inhibition of furin may occur at host/pathogen interface that permits the virus to escape the suppression by mutating key residue that may lead to an altered interface.

Luteolin restricts dengue virus replication through inhibition of the proprotein convertase furin.

In many countries afflicted with dengue fever, traditional medicines are widely used as panaceas for illness, and here we describe the systematic evaluation of a widely known natural product, luteolin, originating from the "heat clearing" class of herbs. We show that luteolin inhibits the replication of all four serotypes of dengue virus, but the selectivity of the inhibition was weak. In addition, ADE-mediated dengue virus infection of human cell lines and primary PBMCs was inhibited. In a time-of-drug-addition study, luteolin was found to reduce infectious virus particle formation, but not viral RNA synthesis, in Huh-7 cells. During the virus life cycle, the host protease furin cleaves the pr moiety from prM protein of immature virus particles in the trans-Golgi network to produce mature virions. Analysis of virus particles from luteolin-treated cells revealed that prM was not cleaved efficiently. Biochemical interrogation of human furin showed that luteolin inhibited the enzyme activity in an uncompetitive manner, with Ki value of 58.6 µM, suggesting that treatment may restrict the virion maturation process. Luteolin also exhibited in vivo antiviral activity in mice infected with DENV, causing reduced viremia. Given the mode of action of luteolin and its widespread source, it is possible that it can be tested in combination with other dengue virus inhibitors.

Bioactive glycosides from the roots of Ilex asprella.

Context The roots of Ilex asprella (Hook. et Arn.) Champ. ex Benth. (Aquifoliaceae) are widely used in Chinese medicine to treat influenza, amygdalitis, pertussis, etc. Their mechanism of action is still unknown, which raises the need to identify new bioactive compounds in this plant. Objective In this study, we isolated a novel saponin containing sulphonic groups, namely, asprellcoside A (1) and a known phenolic glycoside compound (2) from the roots of Ilex asprella and evaluated their bioactivities. Materials and methods Molecular structures were elucidated by analysing their spectral and chemical properties. The viability of A549 cells was tested using a MTT assay. Ability of the compounds to inhibit viruses was determined using the neuraminidase activity assay. Their anti-inflammatory effects were tested using the IP-10 activity assay using various concentrations (compound 1: 0.6, 0.2, 0.6, 1.70, 5.00 and 15.00 µM; compound 2: 0.4, 1.2, 3.6, 11.0, 33.0 and 100 µM). Their inhibitory effect on platelet aggregation induced by adenosine diphosphate (ADP) in rabbit plasma was determined at 60 and 80 µM. Results Both compounds inhibit influenza virus strain A/PuertoRico/8/1934 (H1N1) strongly with EC50 values of 4.1 and 1.7 µM, respectively. Both compounds inhibit the secretion of IP-10 with EC50 values of 6.6 and 2.5 µM, respectively. Compound 1 alone inhibited platelet aggregation significantly, with the rate of suppression being 47 ± 8 and 38 ± 3%, at 60 and 80 µM, respectively. Conclusions The results suggest that both compounds may be valid therapeutics against influenza virus infection and that compound 1 may be a novel agent for treating thrombosis.

Involvement of the azorhizobial chromosome partition gene (parA) in the onset of bacteroid differentiation during Sesbania rostrata stem nodule development.

A parA gene in-frame deletion mutant of Azorhizobium caulinodans ORS571 (ORS571-ΔparA) was constructed to evaluate the roles of the chromosome-partitioning gene on various bacterial traits and on the development of stem-positioned nodules. The ΔparA mutant showed a pleiomorphic cell shape phenotype and was polyploid, with differences in nucleoid sizes due to dramatic defects in chromosome partitioning. Upon inoculation of the ΔparA mutant onto the stem of Sesbania rostrata, three types of immature nodule-like structures with impaired nitrogen-fixing activity were generated. Most showed signs of bacteroid early senescence. Moreover, the ΔparA cells within the nodule-like structures exhibited multiple developmental-stage phenotypes. Since the bacA gene has been considered an indicator for bacteroid formation, we applied the expression pattern of bacA as a nodule maturity index in this study. Our data indicate that the bacA gene expression is parA dependent in symbiosis. The presence of the parA gene transcript was inversely correlated with the maturity of nodule; the transcript was switched off in fully mature bacteroids. In summary, our experimental evidence demonstrates that the parA gene not only plays crucial roles in cellular development when the microbe is free-living but also negatively regulates bacteroid formation in S. rostrata stem nodules.