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Resistance to HER2-targeted therapy represents a significant challenge for the successful treatment of patients with breast cancer with HER2-positive tumors. Through a global mass spectrometry–based proteomics approach, we discovered that the expression of the N6-methyladenosine (m6A) demethylase ALKBH5 was significantly upregulated in HER2-targeted therapy-resistant breast cancer cells. Elevated expression of ALKBH5 was sufficient to confer resistance to HER2-targeted therapy, and specific knockdown of ALKBH5 rescued the efficacy of trastuzumab and lapatinib in resistant breast cancer cells. Mechanistically, ALKBH5 promoted m6A demethylation of GLUT4 mRNA and increased GLUT4 mRNA stability in a YTHDF2-dependent manner, resulting in enhanced glycolysis in resistant breast cancer cells. In breast cancer tissues obtained from patients with poor response to HER2-targeted therapy, increased expression of ALKBH5 or GLUT4 was observed and was significantly associated with poor prognosis in the patients. Moreover, suppression of GLUT4 via genetic knockdown or pharmacologic targeting with a specific inhibitor profoundly restored the response of resistant breast cancer cells to trastuzumab and lapatinib, both in vitro and in vivo. In conclusion, ALKBH5-mediated m6A demethylation of GLUT4 mRNA promotes resistance to HER2-targeted therapy, and targeting the ALKBH5/GLUT4 axis has therapeutic potential for treating patients with breast cancer refractory to HER2-targeted therapies. Significance: GLUT4 upregulation by ALKBH5-mediated m6A demethylation induces glycolysis and resistance to HER2-targeted therapy and represents a potential therapeutic target for treating HER2-positive breast cancer.

Resistance to HER2-targeted therapy represents a significant challenge for the successful treatment of patients with breast cancer with HER2-positive tumors. Through a global mass spectrometry–based proteomics approach, we discovered that the expression of the N6-methyladenosine (m6A) demethylase ALKBH5 was significantly upregulated in HER2-targeted therapy-resistant breast cancer cells. Elevated expression of ALKBH5 was sufficient to confer resistance to HER2-targeted therapy, and specific knockdown of ALKBH5 rescued the efficacy of trastuzumab and lapatinib in resistant breast cancer cells. Mechanistically, ALKBH5 promoted m6A demethylation of GLUT4 mRNA and increased GLUT4 mRNA stability in a YTHDF2-dependent manner, resulting in enhanced glycolysis in resistant breast cancer cells. In breast cancer tissues obtained from patients with poor response to HER2-targeted therapy, increased expression of ALKBH5 or GLUT4 was observed and was significantly associated with poor prognosis in the patients. Moreover, suppression of GLUT4 via genetic knockdown or pharmacologic targeting with a specific inhibitor profoundly restored the response of resistant breast cancer cells to trastuzumab and lapatinib, both in vitro and in vivo. In conclusion, ALKBH5-mediated m6A demethylation of GLUT4 mRNA promotes resistance to HER2-targeted therapy, and targeting the ALKBH5/GLUT4 axis has therapeutic potential for treating patients with breast cancer refractory to HER2-targeted therapies.Significance:GLUT4 upregulation by ALKBH5-mediated m6A demethylation induces glycolysis and resistance to HER2-targeted therapy and represents a potential therapeutic target for treating HER2-positive breast cancer.

Supplementary Data from ALKBH5-Mediated m6A Demethylation of GLUT4 mRNA Promotes Glycolysis and Resistance to HER2-Targeted Therapy in Breast Cancer

Although group 2 Innate Lymphoid Cells (ILC2s) play important roles in driving the pathogenesis of allergic airway inflammation, the molecular mechanisms regulating ILC2 responses remain to be fully elucidated. Adenosine signaling is emerging as an important factor to limit excessive inflammation and tissue damage, its role in ILC2-driven airway inflammation remains to be understood. Here we identify adenosine as a negative regulator of ILC2s and allergic airway inflammation. Elevation of adenosine was observed in lungs after protease papain challenge. Adenosine receptor A2A was abundantly expressed in lung ILC2s. The adenosine analog NECA significantly suppress ILC2s responses and relieved airway inflammation induced by IL-33 or papain. Conversely, blockage of adenosine synthesis by CD73 inhibitor APCP or deficiency of A2A aggravated murine airway inflammation. Adoptive transfer of ILC2s into immunodeficiency NCG mice demonstrated that the regulation of ILC2 by adenosine was cell intrinsic. Mechanistic studies showed that the effects of adenosine on ILC2s were associated with changes in transcriptional profiling, and the elevation of intracellular cAMP and resulted NF-κB downregulation. These observations indicate that adenosine-A2A signaling is a negative regulator of ILC2s, which confers protection against airway inflammation and represents a novel therapeutic target for controlling asthma.

Group 2 innate lymphoid cells (ILC2s) play an important role in allergic airway inflammation. Despite recent advances in defining molecular mechanisms that control ILC2 development and function, the role of endogenous metabolites in the regulation of ILC2s remains poorly understood. Herein, we demonstrated that bilirubin, an end product of heme catabolism, was a potent negative regulator of ILC2s. Bilirubin metabolism was found to be significantly induced during airway inflammation in mouse models. The administration of unconjugated bilirubin (UCB) dramatically suppressed ILC2 responses to interleukin (IL)-33 in mice, including cell proliferation and the production of effector cytokines. Furthermore, UCB significantly alleviated ILC2-driven airway inflammation, which was aggravated upon clearance of endogenous UCB. Mechanistic studies showed that the effects of bilirubin on ILC2s were associated with downregulation of ERK phosphorylation and GATA3 expression. Clinically, newborns with hyperbilirubinemia displayed significantly lower levels of ILC2 with impaired function and suppressed ERK signaling. Together, these findings indicate that bilirubin serves as an endogenous suppressor of ILC2s and might have potential therapeutic value in the treatment of allergic airway inflammation.

Prolonged androgen deprivation therapy (ADT) in patients with prostate cancer can eventually lead to the development of castration-resistant prostate cancer (CRPC). Once CRPC occurs, the patient's prognosis will be extremely poor. This study explored the time to progression and the predictability of risk factors for CRPC progression based on clinical information and laboratory indicators. Among 159 prostate cancer patients initially treated with ADT, 90 patients were screened for inclusion. Patients progressed to CRPC after endocrine therapy enrolled in Group B, and others enrolled in Group A. Within Group B, they were divided into B1 and B2 Group Based on progression to CRPC within 18 months or not. Multi-factor logistic regression analysis showed that the time to PSA nadir (TTN) (P = 0.031) and serum lactate dehydrogenase (LDH) (P = 0.013) were significantly different between Group A and B. TTN (P

COVID-19 has been diffusely pandemic around the world, characterized by massive morbidity and mortality. One of the remarkable threats associated with mortality may be the uncontrolled inflammatory processes, which were induced by SARS-CoV-2 in infected patients. As there are no specific drugs, exploiting safe and effective treatment strategies is an instant requirement to dwindle viral damage and relieve extreme inflammation simultaneously. Here, highly biocompatible glycyrrhizic acid (GA) nanoparticles (GANPs) were synthesized based on GA. In vitro investigations revealed that GANPs inhibit the proliferation of the murine coronavirus MHV-A59 and reduce proinflammatory cytokine production caused by MHV-A59 or the N protein of SARS-CoV-2. In an MHV-A59-induced surrogate mouse model of COVID-19, GANPs specifically target areas with severe inflammation, such as the lungs, which appeared to improve the accumulation of GANPs and enhance the effectiveness of the treatment. Further, GANPs also exert antiviral and anti-inflammatory effects, relieving organ damage and conferring a significant survival advantage to infected mice. Such a novel therapeutic agent can be readily manufactured into feasible treatment for COVID-19.

The roles of RNA modification during organ metastasis of cancer cells are not known. Here we established breast cancer lung metastasis cells by three rounds of selection of lung metastatic subpopulations in vivo and designated them as BCLMF3 cells. In these cells, mRNA N6-methyladenosine (m6A) and methyltransferase METTL3 were increased, while the demethylase FTO was decreased. Epi-transcriptome and transcriptome analyses together with functional studies identified keratin 7 (KRT7) as a key effector for m6A-induced breast cancer lung metastasis. Specifically, increased METTL3 methylated KRT7-AS at A877 to increase the stability of a KRT7-AS/KRT7 mRNA duplex via IGF2BP1/HuR complexes. Furthermore, YTHDF1/eEF-1 was involved in FTO-regulated translational elongation of KRT7 mRNA, with methylated A950 in KRT7 exon 6 as the key site for methylation. In vivo and clinical studies confirmed the essential roles of KRT7, KRT7-AS, and METTL3 for lung metastasis and clinical progression of breast cancer. Collectively, m6A promotes breast cancer lung metastasis by increasing the stability of a KRT7-AS/KRT7 mRNA duplex and translation of KRT7. Significance: This study suggests that N6-methyladenosine is a key driver and potential therapeutic target in breast cancer metastasis.

Group 2 innate lymphoid cells (ILC2s) represent the major player during hyperresponsive airway inflammation. Peroxisome proliferator-activated receptor-γ (PPARγ) was highly expressed on ILC2 and its potential role in asthma has been suggested. However, the detailed mechanism underlying the effects of PPARγ on ILC2-induced airway inflammation remains to be fully understood. Here we identified PPARγ as a positive regulator of lung ILC2. Expression of PPARγ on ILC2 was dramatically induced upon interleukin-33 (IL-33) challenge. Deficiency of PPARγ in hematopoietic system in mice (PPARγfl/fl Vav1Cre) significantly impaired the function of ILC2 in lung, which led to apparent alleviation of airway inflammation in response to IL-33 or Papain challenge, when compared with those in PPARγfl/fl littermates control. Mechanistic studies identified IL-33 receptor ST2 as a transcriptional target of PPARγ. Overexpression of ST2 rescued the functional defects of ILC2 lacking PPARγ. Collectively, these results demonstrated PPARγ as an important regulator of ILC2 during allergic airway inflammation, which sheds new lights on the importance of PPARγ in asthma.

Background tRNA-derived small RNAs (tDRs), which are widely distributed in human tissues including blood and urine, play an important role in the progression of cancer. However, the expression of tDRs in colorectal cancer (CRC) plasma and their potential diagnostic values have not been systematically explored. Methods The expression profiles of tDRs in plasma of CRC and health controls (HCs) are investigated by small RNA sequencing. The level and diagnostic value of 5′-tRF-GlyGCC are evaluated by quantitative PCR in plasma samples from 105 CRC patients and 90 HCs. The mechanisms responsible for biogenesis of 5′-tRF-GlyGCC are checked by in vitro and in vivo models. Results 5′-tRF-GlyGCC is dramatically increased in plasma of CRC patients compared to that of HCs. The area under curve (AUC) for 5′-tRF-GlyGCC in CRC group is 0.882. The combination of carcinoembryonic antigen (CEA) and carbohydrate antigen 199 (CA199) with 5′-tRF-GlyGCC improves the AUC to 0.926. Consistently, the expression levels of 5′-tRF-GlyGCC in CRC cells and xenograft tissues are significantly greater than that in their corresponding controls. Blood cells co-cultured with CRC cells or mice xenografted with CRC tumors show increased levels of 5′-tRF-GlyGCC. In addition, we find that the increased expression of 5′-tRF-GlyGCC is dependent on the upregulation of AlkB homolog 3 (ALKBH3), a tRNA demethylase which can promote tRNA cleaving to generate tDRs. Conclusions The level of 5′-tRF-GlyGCC in plasma is a promising diagnostic biomarker for CRC diagnosis.

N6-methyladenosine (m6A) is the most prevalent RNA modification in eukaryotic mRNAs. Currently available detection methods for locus-specific m6A marks rely on RT-qPCR, radioactive methods, or high-throughput sequencing. Here, we develop a non-qPCR, ultrasensitive, isothermal, and naked-eye visible method for m6A detection based on rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP), named m6A-Rol-LAMP, to verify putative m6A sites in transcripts obtained from the high-throughput data. When padlock probes hybridize to the potential m6A sites on targets, they are converted to circular form by DNA ligase in the absence of m6A modification, while m6A modification hinders the sealing of padlock probes. Subsequently, Bst DNA polymerase-mediated RCA and LAMP allow the amplification of the circular padlock probe to achieve the locus-specific detection of m6A. Following optimization and validation, m6A-Rol-LAMP can ultra-sensitively and quantitatively determine the existence of m6A modification on a specific target site as low as 100 amol under isothermal conditions. Detections of m6A can be performed on rRNA, mRNA, lincRNA, lncRNA and pre-miRNA from biological samples with naked-eye observations after dye incubation. Together, we provide a powerful tool for locus-specific detection of m6A, which can simply, quickly, sensitively, specifically, and visually determine putative m6A modification on RNA.

The outbreak of SARS-CoV-2 lead to a worldwide pandemic which poses substantial challenges to public health.We enrolled 102 consecutive recovered patients with laboratory-confirmed SARS-CoV-2 infection. Epidemiological and demographic characteristics, temporal dynamic profiles of laboratory tests and findings on chest CT radiography, and clinical outcomes were collected and analyzed.Independent risk factors for prolonged fever, viral RNA shedding or radiologic recovery included age of more than 44 years, female gender, having symptoms of cough and fever, a delay from the symptom onset to hospitalization of more than 3 days, a lower CD4 count of less than 500/μL on admission, and severe or critical illness in hospitalization. The estimated median time from symptom onset was 6.4 (5.5 - 7.4) days to peak viral load, 9.1 (7.9 - 10.4) days to afebrile, 8 (6.7 - 9.4) days to worst radiologic finding, 12.7 (11.2 - 14.3) days to viral RNA negativity, and 26.7 (23.8 - 29.9) days to radiologic resolution. This study included the entire cross-section of patients seen in our clinical practice and reflected the real-world situation.These findings provide the rationale for strategies of active symptom monitoring, timing of quarantine and antiviral interventions, and duration of radiologic follow-up in patients with COVID-19.

Gastric cancer (GC) with lymph node metastasis (LNM) at diagnosis is associated with a unstable prognosis and indefinite survival times. The aim of the present study was to construct and validate a model for the Overall survival (OS) estimation for patients with LNM. The nomogram was constructed to predict the OS for LNM-positive GC using the primary group of 836 patients and validated using an independent cohort of 411 patients. Factors in the nomogram were identified by multivariate Cox hazard analysis. The predictive capability of nomogram was evaluated by calibration analysis and decision curve analysis. Multivariate analysis suggested that eight pre-treatment characteristics were used for developing the nomogram. In the primary cohort, the C-index for OS prediction was 0.788 (95% CI: 0.753-0.823), while in validation cohort, the C-index for OS prediction was 0.769 (95% CI: 0. 720-0.818). The calibration plot for the probability of OS and decision curve analyses showed an optimal agreement. Based on the nomogram, we could divided patients into three groups: low-risk group, middle-risk group and a high-risk group(p

Hepatocellular carcinoma (HCC) is the third most frequent cause of cancer-related deaths globally because of high metastasis and recurrence rates. Elucidating the molecular mechanisms of HCC recurrence and metastasis and developing effective targeted therapies are expected to improve patient survival. The promising anti-cancer agents for the treatment of hematological malignancies, histone deacetylase inhibitors (HDIs), have limited effects against epithelial cell-derived cancers, including HCC, the mechanisms involved have not been elucidated. Herein, we studied the molecular mechanisms underlying HDI-induced epithelial-mesenchymal transition (EMT) involving FOXO1-mediated autophagy. Methods: The biological functions of HDIs in combination with autophagy inhibitors were examined both in vitro and in vivo. Cell autophagy was assessed using the generation of mRFP-GFP-LC3-expressing cells and fluorescent LC3 puncta analysis, Western blotting, and electron microscopy. An orthotopic hepatoma model was established in mice for the in vivo experiments. Results: Our study provided novel mechanistic insights into HDI-induced EMT mediated by the autophagy AMPK-FOXO1-ULK1-Snail signaling axis. We demonstrated that autophagy served as a pro-metastasis mechanism in HDI-treated hepatoma cells. HDIs induced autophagy via a FOXO1-dependent pathway, and FOXO1 inhibition promoted HDI-mediated apoptosis in hepatoma cells. Thus, our findings provided novel insights into the molecular mechanisms underlying HDI-induced EMT involving FOXO1-mediated autophagy and demonstrated that a FOXO1 inhibitor exerted a synergistic effect with an HDI to inhibit cell growth and metastasis in vitro and in vivo. Conclusion: We demonstrated that HDIs triggers FOXO1-dependent autophagy, which ultimately promotes EMT, limiting the clinical outcome of HDI-based therapies. Our study suggests that the combination of an HDI and a FOXO1 inhibitor is an effective therapeutic strategy for the treatment of HCC.

Autophagy is a highly conserved catabolic process and participates in a variety of cellular biological activities. The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway, as a critical regulator of autophagy, is involved in the initiation and promotion of a series of pathological disorders including various tumors. Autophagy also participates in regulating the balance between the tumor and the tumor microenvironment. Natural products have been considered a treasure of new drug discoveries and are of great value to medicine. Mounting evidence has suggested that numerous natural products are targeting PI3K/AKT/mTOR-mediated autophagy, thereby suppressing tumor growth. Furthermore, autophagy plays a "double-edged sword" role in different tumors. Targeting PI3K/AKT/mTOR-mediated autophagy is an important therapeutic strategy for a variety of tumors, and plays important roles in enhancing the chemosensitivity of tumor cells and avoiding drug resistance. Therefore, we summarized the roles of PI3K/AKT/mTOR-mediated autophagy in tumorigenesis, progression, and drug resistance of tumors, which may be utilized to design preferably therapeutic strategies for various tumors.

Description, TREM-2 recognizes SARS-CoV-2 membrane protein and activates CD3ζ/ZAP70/STAT1 signal pathway to induce antiviral TH1 response., Limited understanding of T cell responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has impeded vaccine development and drug discovery for coronavirus disease 2019 (COVID-19). We found that triggering receptor expressed on myeloid cells 2 (TREM-2) was induced in T cells in the blood and lungs of patients with COVID-19. After binding to SARS-CoV-2 membrane (M) protein through its immunoglobulin domain, TREM-2 then activated the CD3ζ/ZAP70 complex, leading to STAT1 phosphorylation and T-bet transcription. In vitro stimulation with M protein-reconstituted pseudovirus or recombinant M protein, and TREM-2 promoted the T helper cell 1 (TH1) cytokines interferon-γ and tumor necrosis factor. In vivo infection of CD4–TREM-2 conditional knockout mice with murine coronavirus mouse hepatitis virus A-59 showed that intrinsic TREM-2 in T cells enhanced TH1 response and viral clearance, thus aggravating lung destruction. These findings demonstrate a previously unidentified role for TREM-2 in SARS-CoV-2 infection, and suggest potential strategies for drug discovery and clinical management of COVID-19.

Breast cancer stem cells (BCSCs) are tumor initiating cells that can self-renew and are highly tumorigenic and chemoresistant. Therefore, the identification of factors critical for BCSC function is vital for the development of therapies. Here, we report that DNMT1-mediated FOXO3a promoter hypermethylation leads to downregulation of FOXO3a expression in breast cancer. FOXO3a is functionally related to the inhibition of FOXM1/SOX2 signaling and to the consequent suppression of BCSCs properties and tumorigenicity. Moreover, we found that SOX2 directly transactivates DNMT1 expression and thereby alters the methylation landscape, which in turn feedback inhibits FOXO3a expression. Inhibition of DNMT activity suppressed tumor growth via regulation of FOXO3a/FOXM1/SOX2 signaling in breast cancer. Clinically, we observed a significant inverse correlation between FOXO3a and FOXM1/SOX2/DNMT1 expression levels, and loss of FOXO3a expression or increased expression of FOXM1, SOX2, and DNMT1 predicted poor prognosis in breast cancer. Collectively, our findings suggest an important role of the DNMT1/FOXO3a/FOXM1/SOX2 pathway in regulating BCSCs properties, suggesting potential therapeutic targets for breast cancer.

Targeted inhibition of cyclin-dependent kinase 7 (CDK7) via its covalent inhibitor THZ1 can suppress the growth of various cancers, while its roles on colorectal cancer (CRC) remain obscure. Here we report that the expression of CDK7 is upregulated in CRC cells and tissues. THZ1 exhibits high potency and selectivity against CRC cells both in vitro and in vivo via induction of cell apoptosis rather than cell cycle disruption. Intriguingly, THZ1 treatment increases the ability of epithelial mesenchymal transition (EMT) and in vivo metastasis to liver of CRC cells. Mechanistical studies reveal that THZ1 increases the expression of Snail, while not other EMT-transcription factors, via enhancing its protein stability rather than mRNA expression or translation. By screening Snail stability related factors via qRT-PCR, results indicate THZ1 and si-CDK7 decrease the expression of protein kinase D1 (PKD1) in CRC cells. Down regulation of PKD1 mediates THZ1 up regulated Snail via dephosphorylation of Snail Ser 11 and prevention of proteasome mediated degradation. Clinical analysis confirms that CDK7 is significantly (p

Background: Gut ecosystem has profound effects on host physiology and health. Gastrointestinal (GI) symptoms were frequently observed in patients with COVID-19. Compared with other organs, gut antiviral response can result in more complicated immune responses because of the interactions between the gut microbiota and host immunity. However, there are still large knowledge gaps in the impact of COVID-19 on gut molecular profiles and commensal microbiome, hindering our comprehensive understanding of the pathogenesis of SARS-CoV-2 and the treatment of COVID-19.Results: We performed longitudinal stool multi-omics profiling to systemically investigate the molecular phenomics alterations of gut ecosystem in COVID-19. Gut proteomes of COVID-19 were characterized by disturbed immune, proteolysis and redox homeostasis. The expression and glycosylation of proteins involved in neutrophil degranulation and migration were suppressed, while those of proteases were upregulated. The variable domains of Ig heavy chains were downregulated and the overall glycosylation of IgA heavy chain constant regions, IgGFc-binding protein, and J chain were suppressed with glycan-specific variations. There was a reduction of beneficial gut bacteria and an enrichment of bacteria derived deleterious metabolites potentially associated with multiple types of diseases (such as ethyl glucuronide). The reduction of Ig heave chain variable domains may contribute to the increase of some Bacteroidetes species. Many bacteria ceramide lipids with a C17-sphingoid based were downregulated in COVID-19. In many cases, the gut phenome did not restore two months after symptom onset.Conclusions: Our study indicates widely disturbed gut molecular profiles which may play a role in the development of symptoms in COVID-19. Our findings also emphasis the need for ongoing investigation of the long-term gut molecular and microbial alterations during COVID-19 recovery process. Considering the gut ecosystem as a potential target could offer a valuable approach in managing the disease.

Additional file 1: Fig. S1. The standard curve of 5′-tRF-GlyGCC quantification. Fig. S2. ALKBH3 is involved in the biogenesis of 5′-tRF-GlyGCC. Fig. S3. The nude mice (A) and BALB/c (B) mice bearing CRC cells xenografted tumor. Table S1. Background information of the Small RNA sequencing samples. Table S2. Background demographic of the study cohorts. Table S4. Relationship between the levels of 5′-tRF-GlyGCC and the clinicopathological variables in CRC patients. Table S5. Clinical diagnosis utility about various marker alone and their combination effects for CRC diagnosis.

Although human antibodies elicited by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein are profoundly boosted upon infection, little is known about the function of N-reactive antibodies. Herein, we isolated and profiled a panel of 32 N protein-specific monoclonal antibodies (mAbs) from a quick recovery coronavirus disease-19 (COVID-19) convalescent patient who had dominant antibody responses to the SARS-CoV-2 N protein rather than to the SARS-CoV-2 spike (S) protein. The complex structure of the N protein RNA binding domain with the mAb with the highest binding affinity (nCoV396) revealed changes in the epitopes and antigen’s allosteric regulation. Functionally, a virus-free complement hyper-activation analysis demonstrated that nCoV396 specifically compromises the N protein-induced complement hyper-activation, which is a risk factor for the morbidity and mortality of COVID-19 patients, thus laying the foundation for the identification of functional anti-N protein mAbs.

Although human antibodies elicited by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein are profoundly boosted upon infection, little is known about the function of N-reactive antibodies. Herein, we isolate and profile a panel of 32 N protein-specific monoclonal antibodies (mAbs) from a quick recovery coronavirus disease-19 (COVID-19) convalescent patient who has dominant antibody responses to the SARS-CoV-2 N protein rather than to the SARS-CoV-2 spike (S) protein. The complex structure of the N protein RNA binding domain with the highest binding affinity mAb (nCoV396) reveals changes in the epitopes and antigen’s allosteric regulation. Functionally, a virus-free complement hyperactivation analysis demonstrates that nCoV396 specifically compromises the N protein-induced complement hyperactivation, which is a risk factor for the morbidity and mortality of COVID-19 patients, thus laying the foundation for the identification of functional anti-N protein mAbs.

Although human antibodies elicited by severe acute respiratory distress syndrome coronavirus-2 (SARS-CoV-2) nucleocapsid (N) protein are profoundly boosted upon infection, little is known about the function of N-directed antibodies. Herein, we isolated and profiled a panel of 32 N protein-specific monoclonal antibodies (mAb) from a quick recovery coronavirus disease-19 (COVID-19) convalescent, who had dominant antibody responses to SARS-CoV-2 N protein rather than to Spike protein. The complex structure of N protein RNA binding domain with the highest binding affinity mAb nCoV396 reveals the epitopes and antigen’s allosteric changes. Functionally, a virus-free complement hyper-activation analysis demonstrates that nCoV396 specifically compromises N protein-induced complement hyper-activation, a risk factor for morbidity and mortality in COVID-19, thus paving the way for functional anti-N mAbs identification.One Sentence SummaryB cell profiling, structural determination, and protease activity assays identify a functional antibody to N protein.

BackgroundEffective therapies are urgently needed for the SARS-CoV-2 pandemic. Chloroquine has been proved to have antiviral effect against coronavirus in vitro. In this study, we aimed to assess the efficacy and safety of chloroquine with different doses in COVID-19.MethodIn this multicenter prospective observational study, we enrolled patients older than 18 years old with confirmed SARS-CoV-2 infection excluding critical cases from 12 hospitals in Guangdong and Hubei Provinces. Eligible patients received chloroquine phosphate 500mg, orally, once (half dose) or twice (full dose) daily. Patients treated with non-chloroquine therapy were included as historical controls. The primary endpoint is the time to undetectable viral RNA. Secondary outcomes include the proportion of patients with undetectable viral RNA by day 10 and 14, hospitalization time, duration of fever, and adverse events.ResultsA total of 197 patients completed chloroquine treatment, and 176 patients were included as historical controls. The median time to achieve an undetectable viral RNA was shorter in chloroquine than in non-chloroquine (absolute difference in medians −6.0 days; 95% CI −6.0 to −4.0). The duration of fever is shorter in chloroquine (geometric mean ratio 0.6; 95% CI 0.5 to 0.8). No serious adverse events were observed in the chloroquine group. Patients treated with half dose experienced lower rate of adverse events than with full dose.ConclusionsAlthough randomised trials are needed for further evaluation, this study provides evidence for safety and efficacy of chloroquine in COVID-19 and suggests that chloroquine can be a cost-effective therapy for combating 102 the COVID-19 pandemic.

Objectives To retrospectively analyze the most common imaging features on CT at baseline and as they evolve with time as the disease progresses or resolves in a cohort of patients affected with 2019 coronal virus disease (COVID-19) pneumonia in Zhuhai, China.Methods We evaluated 38 patients with COVID-19 in the authors’ institution from Jan 1 to Jan 31, 2020. Cases were confirmed by real-time RT-PCR and were analyzed for epidemiological, demographic, clinical, and radiological features. Outcomes were followed up until Feb 18, 2020. Results 38 initial scans and 62 follow-up scans were obtained. 28 (74%) patients had the history of travel to or residence in Hubei Province of China in 14 days prior to the illness onset. Common findings included ground-glass opacification (GGO), sometimes mixed with consolidation, and interlobular septal and intralobular interstitial thickening. Follow-up imaging often demonstrated peripheral GGO and consolidations spreading to the remainder of the lungs and the increasing consolidative component reflecting the progression of the disease. 8 patients (21%) whose swabs or serum were positive for COVID-19 had no imaging findings on CT throughout the disease course. After treatment the serum and sputum tests became negative for COVID-19 in 32(84%) cases. 28(74%) patients were discharged and three (8%) of them were transferred to the Observation Ward, while seven (18%) patients were kept in Isolation Ward. Conclusion The commonest pattern observed was GGO alone or GGO mixed with consolidation predominantly in lower and peripheral lungs. The follow-up CT scan is crucial for the diagnosis and evaluation of the disease process.

Background: Coronavirus Disease 2019 (COVID-19) caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global threat to public health. Aiming to construct an efficient screening pattern, we comprehensively evaluated the performances of RT-PCR and chest CT in diagnosing COVID-19. Methods: The records including demographics, RT-PCR, and CT from 87 confirmed COVID-19 cases and 481 exclusion cases were collected. The diagnostic accuracy of the pharyngeal swab RT-PCR, CT, combination with the second pharyngeal swab RT-PCR or with CT were evaluated individually. Besides, all the stool RT-PCR results were plotted by time to explore the value of stool RT-PCR. Findings: Combination of RT-PCR and CT has the higher sensitivity (91.9%,79/86) than RT-PCR alone (78.2%,68/87) or CT alone (66.7%, 54 of 81) or combination of two RT-PCR tests (86.2%,75/87). There was good agreement between RT-PCR and CT (kappa-value, 0.430). In 34 COVID-19 cases with inconsistent results, 94.1% (n=32) are mild infection, 62.5% of which (20/32) showed positive RT-PCR. 46.7% (35/75) COVID-19 patients had at least one positive stool during the course. Two cases had positive stool earlier than the pharyngeal swabs. Importantly, one patient had consecutive positive stool but negative pharyngeal swabs. Interpretation: Combination of RT-PCR and CT with the highest sensitivity is an optimal pattern to screen COVID-19. RT-PCR is superior to CT in diagnosing mild infections. Stool RT-PCR should be considered as an item for improving discovery rate and hospital discharge. This study shed light for optimizing scheme of screening and monitoring of SARS-CoV-2 infection. Funding Statement: This work was supported by the National Natural Science Foundation of China (No. 81502104), National Program on Key Basic Research Project (No. 2018YFC0910600), the Nature Science Foundation of Guangdong Province, China (Grant No: 2017A030313771 and 2020A151501001 ) and the Young Teachers Nurturing Program of Sun Yat-Sen University (Grant No:17ykpy62). Declaration of Interests: All authors declare no competing interests. Ethics Approval Statement: The study was approved by The Fifth Affiliated Hospital of Sun Yat-sen University Ethics Committee and written informed consent was obtained from patients involved before enrolment when data were collected retrospectively.

The new coronavirus (SARS-CoV-2) outbreak originating from Wuhan, China, poses a threat to global health. While it’s evident that the virus invades respiratory tract and transmits from human to human through airway, other viral tropisms and transmission routes remain unknown. We tested viral RNA in stool from 73 SARS-CoV-2-infected hospitalized patients using rRT-PCR. 53.42% of the patients tested positive in stool. 23.29% of the patients remained positive in feces even after the viral RNA decreased to undetectable level in respiratory tract. The viral RNA was also detected in gastrointestinal tissues. Furthermore, gastric, duodenal and rectal epithelia showed positive immunofluorescent staining of viral host receptor ACE2 and viral nucleocapsid protein in a case of SARS-CoV-2 infection. Our results provide evidence for gastrointestinal infection of SARS-CoV-2, highlighting its potential fecal-oral transmission route.

Nowadays, risk factors of triple-negative breast cancer (TNBC) metastasis are not well identified. Our present study reveals that an industrial chemical, bisphenol S (BPS), can promote the migration, but not the proliferation, of TNBC cells in vitro. BPS activates YAP, a key effector of Hippo pathway, by inhibiting its phosphorylation, which promotes YAP nuclear accumulation and up-regulates its downstream genes such as CTGF and ANKRD1. Inhibition of YAP blocks the BPS-triggered cell migration and up-regulation of fibronectin (FN) and vimentin (Vim). BPS rapidly decreases the phosphorylation levels of LATS1 (Ser909) in TNBC cells, which regulates the activation and functions of YAP. Silencing LATS1/2 by siRNA increases BPS-induced dephosphorylation of YAP and extended the half-life of YAP protein. Inhibition of G protein-coupled estrogen receptor 1 (GPER) and its downstream PLCβ/PKC signals attenuate the effects of BPS-induced YAP dephosphorylation and CTGF up-regulation. Targeted inhibition of GPER/YAP inhibits BPS-induced migration of TNBC cells. Collectively, we reveal that GPER/Hippo-YAP signal is involved in BPS-induced migration of TNBC cells.

Hepatocellular carcinoma (HCC) remains the third cause of cancer-related mortality. Resection and transplantation are the only curative treatments available but are greatly hampered by high recurrence rates and development of metastasis, the initiation of cancer metastasis requires migration and invasion of cells, which is enabled by epithelial-mesenchymal transitions (EMT). TGF-β1 is a secreted protein that performs many cellular functions, including the control of cell growth, cell proliferation, cell differentiation and apoptosis. TGF-β1 is known as a major inducer of EMT, and it was reported that TGF-β1 induced EMT via Smad-dependent and Smad-independent pathways. However, the extrinsic signals of TGF-β1 regulated the EMT in hepatoma cells remains to be elucidated, and searching drugs to inhibit TGF-β1 induced EMT may be considered to be a potentially effective therapeutic strategy in HCC. Fortunately, in this study, we found that curcumin inhibited TGF-β1-induced EMT in hepatoma cells. Furthermore, we demonstrated that curcumin inhibited TGF-β1-induced EMT via inhibiting Smad2 phosphorylation and nuclear translocation, then suppressing Smad2 combined with the promoter of Snail which inhibited the transcriptional expression of Snail. These findings suggesting curcumin could be a useful agent for antitumor therapy and also a promising drug combined with other strategies to preventing and treating HCC.

Gut ecosystem has profound effects on host physiology and health. Gastrointestinal (GI) symptoms were frequently observed in patients with COVID-19. Compared with other organs, gut antiviral response can result in more complicated immune responses because of the interactions between the gut microbiota and host immunity. However, there are still large knowledge gaps in the impact of COVID-19 on gut molecular profiles and commensal microbiome, hindering our comprehensive understanding of the pathogenesis of SARS-CoV-2 and the treatment of COVID-19. We performed longitudinal stool multi-omics profiling to systemically investigate the molecular phenomics alterations of gut ecosystem in COVID-19. Gut proteomes of COVID-19 were characterized by disturbed immune, proteolysis and redox homeostasis. The expression and glycosylation of proteins involved in neutrophil degranulation and migration were suppressed, while those of proteases were upregulated. The variable domains of Ig heavy chains were downregulated and the overall glycosylation of IgA heavy chain constant regions, IgGFc-binding protein, and J chain were suppressed with glycan-specific variations. There was a reduction of beneficial gut bacteria and an enrichment of bacteria derived deleterious metabolites potentially associated with multiple types of diseases (such as ethyl glucuronide). The reduction of Ig heave chain variable domains may contribute to the increase of some Bacteroidetes species. Many bacteria ceramide lipids with a C17-sphingoid based were downregulated in COVID-19. In many cases, the gut phenome did not restore two months after symptom onset. Our study indicates widely disturbed gut molecular profiles which may play a role in the development of symptoms in COVID-19. Our findings also emphasis the need for ongoing investigation of the long-term gut molecular and microbial alterations during COVID-19 recovery process. Considering the gut ecosystem as a potential target could offer a valuable approach in managing the disease., Graphical abstract Image 1

Background Estrogenic signals are suggested to have protection roles in the development of colorectal cancer (CRC). The G protein-coupled estrogen receptor (GPER) has been reported to mediate non-genomic effects of estrogen in hormone related cancers except CRC. Its expression and functions in CRC were investigated. Methods The expression of GPER and its associations with clinicopathological features were examined. The mechanisms were further investigated using cells, mouse xenograft models, and clinical human samples. Results GPER was significantly (p

Triple-negative breast cancer (TNBC) is characterized by high vascularity and frequent metastasis. Here, we found that activation of G protein-coupled estrogen receptor (GPER) by its specific agonist G-1 can significantly inhibit interleukin 6 (IL-6) and vascular endothelial growth factor A (VEGF-A). TNBC tissue microarrays from 100 TNBC patients revealed GPER is negatively associated with IL-6 levels and higher grade and stage. Activation of GPER or anti-IL-6 antibody can inhibit both in vitro tube formation of human umbilical vein endothelial cells (HUVECs) and migration of TNBC cells. While recombinant IL-6 supplementary can significantly reverse the inhibitory effects of G-1, suggesting the essential role of IL-6 in G-1 induced suppression of angiogenesis and invasiveness of TNBC cells. G-1 treatment decreased the phosphorylation, nuclear localization, transcriptional activities of NF-κB and suppressed its binding with IL-6 promoter. BAY11-7028, the inhibitor of NF-κB, can mimic the effect of G-1 to suppression of IL-6 and VEGF-A. While over expression of p65 can attenuate the inhibitory effects of G-1 on IL-6 and VEGF expression. The suppression of IL-6 by G-1 can further inhibit HIF-1α and STAT3 signals in TNBC cells by inhibition their expression, phosphorylation and/or nuclear localization. Moreover, G-1 also inhibited the in vivo NF-κB/IL-6 signals and angiogenesis and metastasis of MDA-MB-231 xenograft tumors. In conclusion, our study demonstrated that activation of GPER can suppress migration and angiogenesis of TNBC via inhibition of NF-κB/IL-6 signals, therefore it maybe act as an important target for TNBC treatment.

Radiotherapy is the primary treatment for nasopharyngeal carcinoma (NPC). Patients with intermediate and advanced stage NPC receiving only radiotherapy have limited survival, so newer immunotherapeutic approaches are sought. The major impediment to better clinical outcomes is tumor immune tolerance. Indoleamine 2,3-dioxygenase (IDO), an IFNγ-inducible enzyme, is a major inducer of immune tolerance during tumor development; therefore, inhibition of the IDO pathway is an important modality for cancer treatment. We show that bortezomib, a proteasomal inhibitor, inhibited the pathways leading to STAT1 and IRF-1 activation, both of which are necessary for IDO expression. Bortezomib downregulated IFNγ-induced IDO expression via inhibition of STAT1 phosphorylation and nuclear translocation, thereby suppressing STAT1-driven IDO transcription in NPC cells. Bortezomib also promoted IκB-α phosphorylation-ubiquitination, which released NF-κB from IκB-α. However, the released NF-κB could not enter the nucleus to conduct its biological effects and accumulated in the cytoplasm. Negative feedback inhibited the transcription of NF-κB, which is important for activating IRF-1 expression. IDO expression is regulated by two important transcription factor binding sites, ISREs, which bind STAT1 and IRF-1, and GASs, which binds STAT1. Bortezomib upregulated IRF-1 protein by inhibiting its proteasome-dependent degradation, but it also inhibited STAT1 phosphorylation, which directly inhibited the activation of GAS and indirectly inhibited the activation of ISRE, which needs both STAT1 and IRF-1. These discoveries provide a mechanism for the antitumor action of bortezomib and have implications for the development of clinical cancer immunotherapy for preventing and treating NPC. Cancer Immunol Res; 5(1); 42–51. ©2016 AACR.

Autophagy is a highly conserved catabolic process that mediates degradation of pernicious or dysfunctional cellular components, such as invasive pathogens, senescent proteins, and organelles. It can promote or suppress tumor development, so it is a “double-edged sword” in tumors that depends on the cell and tissue types and the stages of tumor. The epithelial-mesenchymal transition (EMT) is a complex biological trans-differentiation process that allows epithelial cells to transiently obtain mesenchymal features, including motility and metastatic potential. EMT is considered as an important contributor to the invasion and metastasis of cancers. Thus, clarifying the crosstalk between autophagy and EMT will provide novel targets for cancer therapy. It was reported that EMT-related signal pathways have an impact on autophagy; conversely, autophagy activation can suppress or strengthen EMT by regulating various signaling pathways. On one hand, autophagy activation provides energy and basic nutrients for EMT during metastatic spreading, which assists cells to survive in stressful environmental and intracellular conditions. On the other hand, autophagy, acting as a cancer-suppressive function, is inclined to hinder metastasis by selectively down-regulating critical transcription factors of EMT in the early phases. Therefore, the inhibition of EMT by autophagy inhibitors or activators might be a novel strategy that provides thought and enlightenment for the treatment of cancer. In this article, we discuss in detail the role of autophagy and EMT in the development of cancers, the regulatory mechanisms between autophagy and EMT, the effects of autophagy inhibition or activation on EMT, and the potential applications in anticancer therapy.

Background Long noncoding RNAs (lncRNAs) have emerged as critical players in cancer progression, but their functions in colorectal cancer (CRC) metastasis have not been systematically clarified. Methods lncRNA expression profiles in matched normal and CRC tissue were checked using microarray analysis. The biological roles of a novel lncRNA, namely RP11-138 J23.1 (RP11), in development of CRC were checked both in vitro and in vivo. Its association with clinical progression of CRC was further analyzed. Results RP11 was highly expressed in CRC tissues, and its expression increased with CRC stage in patients. RP11 positively regulated the migration, invasion and epithelial mesenchymal transition (EMT) of CRC cells in vitro and enhanced liver metastasis in vivo. Post-translational upregulation of Zeb1, an EMT-related transcription factor, was essential for RP11-induced cell dissemination. Mechanistically, the RP11/hnRNPA2B1/mRNA complex accelerated the mRNA degradation of two E3 ligases, Siah1 and Fbxo45, and subsequently prevented the proteasomal degradation of Zeb1. m6A methylation was involved in the upregulation of RP11 by increasing its nuclear accumulation. Clinical analysis showed that m6A can regulate the expression of RP11, further, RP11 regulated Siah1-Fbxo45/Zeb1 was involved in the development of CRC. Conclusions m6A-induced lncRNA RP11 can trigger the dissemination of CRC cells via post-translational upregulation of Zeb1. Considering the high and specific levels of RP11 in CRC tissues, our present study paves the way for further investigations of RP11 as a predictive biomarker or therapeutic target for CRC.