Identification of benzo[b]thiophene-1,1-dioxide derivatives as novel PHGDH covalent inhibitors.

The increased de novo serine biosynthesis confers many advantages for tumorigenesis and metastasis. Phosphoglycerate dehydrogenase (PHGDH), a rate-limiting enzyme in serine biogenesis, exhibits hyperactivity across multiple tumors and emerges as a promising target for cancer treatment. Through screening our in-house compound library, we identified compound Stattic as a potent PHGDH inhibitor (IC50 = 1.98 ± 0.66 µM). Subsequent exploration in structural activity relationships led to the discovery of compound B12 that demonstrated the increased enzymatic inhibitory activity (IC50 = 0.29 ± 0.02 µM). Furthermore, B12 exhibited robust inhibitory effects on the proliferation of MDA-MB-468, NCI-H1975, HT1080 and PC9 cells that overexpress PHGDH. Additionally, using a [U-13C6]-glucose tracing assay, B12 was found to reduce the production of glucose-derived serine in MDA-MB-468 cells. Finally, mass spectrometry-based peptide profiling, mutagenesis experiment and molecular docking study collectively suggested that B12 formed a covalent bond with Cys421 of PHGDH.

Identifying the pathophysiological traits of obstructive sleep apnea during dexmedetomidine sedation.

Dexmedetomidine (DEX) has been described as a safe sedative in clinical practice, but its effects on the pathophysiological traits of obstructive sleep apnea (OSA) are unclear. We estimated the effects of DEX sedation on the four key pathophysiological traits of OSA (pharyngeal collapsibility, dilator muscle function, arousal threshold, and loop gain) in adult patients with OSA by conducting a secondary analysis of a prospective diagnostic trial. Pathophysiological traits estimated from polysomnography and the respiratory parameters under natural sleep and DEX-induced sleep were compared. Bivariate and multivariate linear regression analyses were used to estimate the relationship between pathophysiological traits and OSA severity for both sleep states. Adult patients with OSA had a significantly higher pharyngeal collapsibility (Vpassive : 44.9 [15.7 to 53.8] vs. 53.3 [34.2 to 66.3] %eupnea , p < 0.001), arousal threshold (178.5 [132.5 to 234.6] vs. 140.5 [123.2 to 192.3] %eupnea , p < 0.001), and loop gain (LG1: 0.74 ± 0.25 vs. 0.60 ± 0.17, p < 0.001; LGn: 0.52 ± 0.12 vs. 0.44 ± 0.08, p < 0.001) during DEX-induced sleep compared with natural sleep. There was no significant difference in dilator muscle function or PSG respiratory parameters between natural versus DEX-induced sleep states. Bivariate regression analysis showed varying degrees of correlation between OSA traits and severity. Multiple regression analysis indicated that collapsibility was the strongest predictor of the apnea-hypopnea index for both sleep states. Dexmedetomidine sedation in patients with OSA increased the pharyngeal collapsibility without impairing dilator muscle function, while elevating arousal threshold and increasing loop gain.

SUMO specific peptidase 3 halts pancreatic ductal adenocarcinoma metastasis via deSUMOylating DKC1.

In the past few decades, advances in the outcomes of patients suffering from pancreatic ductal adenocarcinoma (PDAC) have lagged behind these gained in the treatment of many other malignancies. Although the pivotal role of the SUMO pathway in PDAC has been illustrated, the underlying molecule drivers have yet to be fully elucidated. In the present study, we identified SENP3 as a potential suppressor of PDAC progression through an in vivo metastatic model. Further studies revealed that SENP3 inhibited PDAC invasion in a SUMO system dependent fashion. Mechanistically, SENP3 interacted with DKC1 and, as such, catalyzed the deSUMOylation of DKC1, which accepted SUMO3 modifiers at three lysine residues. SENP3-mediated deSUMOylation caused DKC1 instability and disruption of the interaction between snoRNP proteins, which contributed to the impaired migration ability of PDAC. Indeed, overexpression of DKC1 abated the anti-metastasis effect of SENP3, and DKC1 was elevated in PDAC specimens and associated with a poor prognosis in PDAC patients. Collectively, our findings shed light on the essential role of SENP3/DKC1 axis in the progression of PDAC.

Case report: Image guidance retrieval of a foreign body in retropharyngeal space.

Background: Ingested foreign bodies fully embedded in retropharyngeal space present a unique challenge, as they can be difficult to locate and visualize via classic transoral laryngoscopy or the transcervical approach. Methods: We retrieved a complete extraluminal chicken bone located in the patient's retropharyngeal space at the level of the C4-C5 spine through a well-designed transcervical approach with a combination of image-guided neck navigation. Conclusion: A combined use of image-guided neck navigation and a dedicated transcervical approach for location of a foreign body in the retropharyngeal space is practical and available for clinical application.

Pregnancy outcomes in different stages of systemic lupus erythematosus among Chinese women - a retrospective cohort study.

OBJECTIVES: To analyze the outcomes of pregnancies and risk factors in Chinese women with different stages of systemic lupus erythematosus (SLE). MATERIAL AND METHODS: A total of 55 conceptions in 52 patients with SLE between Jan 2007 and Jan 2019 were retrospected systematically from a general hospital graded 3A in China. Medical records provided us a good way to retrieve the clinical parameters and lab data of patients. RESULTS: Pregnant women with SLE activity had significant hyperimmunoglobulin, hypocomplement, low platelet counts, high erythrocyte sedimentation rate, C-reactive protein and 24-h urine protein. Hydroxychloroquine had been used to reduce the rates of SLE activity in pregnant women. Logistic regression analysis showed low platelet counts, hypocomplement and 24-h urine protein were significantly correlated with fetal loss. Compared to those in stable stage, the active SLE patients have more risks of hypertensive disorders of pregnancy, thrombocytopenia, lupus nephritis and placental infarction, and have worse fetal outcomes, including the higher rate of fetal loss, preterm and asphyxia neonatorum. CONCLUSIONS: Different stages of SLE during pregnancy are closely related to maternal and fetal outcomes. It is imperative to provide SLE women with pregnancy consultation and regular multispecialty care.

The interval between onset and admission predicts disease progression in COVID-19 patients.

BACKGROUND: The prognostic role of the interval between disease onset and hospital admission (O-A interval) was undetermined in patients with the coronavirus disease 2019 (COVID-19). METHODS: A total of 205 laboratory-confirmed inpatients admitted to Hankou hospital of Wuhan from January 11 to March 8, 2020 were consecutively included in this retrospective observational study. Demographic data, medical history, laboratory testing results were collected from medical records. Univariate and multivariate logistic regression models were used to evaluate the prognostic effect of the O-A interval (≤7 versus >7 days) on disease progression in mild-to-moderate patients. For severe-to-critical patients, the in-hospital mortality and the length of hospital stay were compared between the O-A interval subgroups using log-rank test and Mann-Whitney U test, respectively. RESULTS: Mild-to-moderate patients with a short O-A interval (≤7 days) are more likely to deteriorate to severe-to-critical stage compared to those with a long O-A interval (>7 days) [unadjusted odds ratio =2.93, 95% confidence interval (CI), 1.32-6.55; adjusted odds ratio =3.44, 95% CI, 1.20-9.83]. No association was identified between the O-A interval and the mortality or the length of hospital stay of severe-to-critical patients. CONCLUSIONS: The O-A interval has predictive values for the disease progression in mild-to-moderate COVID-19 patients. Under circumstances of the specific health system in Wuhan, China, the spontaneous healthcare-seeking behavior is usually determined by patients' own heath conditions. Hence, the O-A interval can be reflective of the natural course of COVID-19 to some extent. However, our findings should be validated further in other cohorts and in other health systems.

The deubiquitinase USP44 promotes Treg function during inflammation by preventing FOXP3 degradation.

The transcription factor forkhead box P3 (FOXP3) is essential for the development of regulatory T cells (Tregs) and their function in immune homeostasis. Previous studies have shown that in natural Tregs (nTregs), FOXP3 can be regulated by polyubiquitination and deubiquitination. However, the molecular players active in this pathway, especially those modulating FOXP3 by deubiquitination in the distinct induced Treg (iTreg) lineage, remain unclear. Here, we identify the ubiquitin-specific peptidase 44 (USP44) as a novel deubiquitinase for FOXP3. USP44 interacts with and stabilizes FOXP3 by removing K48-linked ubiquitin modifications. Notably, TGF-ß induces USP44 expression during iTreg differentiation. USP44 co-operates with USP7 to stabilize and deubiquitinate FOXP3. Tregs genetically lacking USP44 are less effective than their wild-type counterparts, both in vitro and in multiple in vivo models of inflammatory disease and cancer. These findings suggest that USP44 plays an important role in the post-translational regulation of Treg function and is thus a potential therapeutic target for tolerance-breaking anti-cancer immunotherapy.

The Long Non-Coding RNA MALAT1 Enhances Ovarian Cancer Cell Stemness by Inhibiting YAP Translocation from Nucleus to Cytoplasm.

BACKGROUND The purpose of this work was to unearth the effects and underlying mechanism of long non-coding RNA (lncRNA) MALAT1 in ovarian cancer cell stemness. MATERIAL AND METHODS Western blot, quantitative polymerase chain reaction (qPCR) and sphere forming analysis were performed to evaluate the stem-like traits of cells and MALAT1-induced effects on ovarian cancer cell stemness. Cell viability was performed to evaluate MALAT1 role in the chemoresistance of ovarian cancer cells. RNA immunoprecipitation (RIP) and luciferase reporter analysis were constructed to investigate the underlying mechanisms. RESULTS Here, qPCR assay showed that MALAT1 level was remarkably higher in non-adherent spheres formed by adherent ovarian cancer cells, as well as cisplatin-resistant ovarian cancer cells. Additionally, MALAT1 knockdown reduced ovarian cancer cell stemness, characterized as the decrease of sphere forming ability, expression of stemness regulatory masters, and attenuation of cisplatin resistance. Moreover, MALAT1 interacted with yes-associated protein (YAP), inhibited its nuclear-cytoplasm translocation, promoted YAP protein stability and expression and thus increased its activity. Notably, rescuing expression of YAP attenuated the inhibition of MALAT1 knockdown on ovarian cancer cell stemness. CONCLUSIONS In conclusion, these results demonstrate a MALAT1/YAP axis responsible for ovarian cancer cell stemness.

RNA Binding Protein RNPC1 Suppresses the Stemness of Human Endometrial Cancer Cells via Stabilizing MST1/2 mRNA.

BACKGROUND RNA binding protein RNPC1 has a tumor-suppressive role in various tumors, nevertheless, the role of RNPC1 in human endometrial cancer (EC) are never been reported. MATERIAL AND METHODS Western blot, quantitative polymerase chain reaction and sphere forming analysis were performed to evaluate the stem-like traits of cells and RNPC1-induced effects on EC cell stemness. RNA immunoprecipitation (RIP) was constructed to investigate the underlying mechanisms. RESULTS The spheres formed by EC cells, named EC spheres, exhibited a remarkably higher stemness than the parental cells, which is characterized as the increase of sphere forming ability, ALDH1 activity, stemness marker expression and migration ability. Notably, RNPC1 expression was decreased in poorly differentiated EC cells than that in EC cells with moderately differentiated. Additionally, RNPC1 expression was significantly decreased in EC spheres and RNPC1 overexpression attenuated the stemness of EC spheres. Moreover, RNPC1 overexpression decreased the migration ability of EC spheres. Mechanistic studies showed that RNPC1 overexpression activated the Hippo pathway through directly binding to MST1/2. Inhibition of MST1/2 rescued RNPC1-mediated effects on EC sphere stemness. CONCLUSIONS Therefore, our results indicate a novel RNPC1/MST1/2 signaling responsible for EC cell stemness.

Nemo-like Kinase Drives Foxp3 Stability and Is Critical for Maintenance of Immune Tolerance by Regulatory T Cells.

The Foxp3 transcription factor is a crucial determinant of both regulatory T (TREG) cell development and their functional maintenance. Appropriate modulation of tolerogenic immune responses therefore requires the tight regulation of Foxp3 transcriptional output, and this involves both transcriptional and post-translational regulation. Here, we show that during T cell activation, phosphorylation of Foxp3 in TREG cells can be regulated by a TGF-ß activated kinase 1 (TAK1)-Nemo-like kinase (NLK) signaling pathway. NLK interacts and phosphorylates Foxp3 in TREG cells, resulting in the stabilization of protein levels by preventing association with the STUB1 E3-ubiquitin protein ligase. Conditional TREG cell NLK-knockout (NLKΔTREG) results in decreased TREG cell-mediated immunosuppression in vivo, and NLK-deficient TREG cell animals develop more severe experimental autoimmune encephalomyelitis. Our data suggest a molecular mechanism, in which stimulation of TCR-mediated signaling can induce a TAK1-NLK pathway to sustain Foxp3 transcriptional activity through the stabilization of protein levels, thereby maintaining TREG cell suppressive function.

Poly-functional T helper cells in human tonsillar mononuclear cells.

Tonsils are important lymphoid organs in which B cells and T cells complete their maturation and identify cells that are infected by pathogens. However, the functions of T cells in human tonsils remain unclear, especially the characteristics of polyfunctional CD4+ T helper cells. In this study, we used multi-color flow cytometry to analyze the expression or co-expression of effector cytokines in CD4+ T cells from tonsillar tissues. We have demonstrated that tonsillar CD4+ T cell can express various Th effector cytokines after short-term polyclonal stimulation, and that cytokine-producing CD4+ T cells were CD45RO+ T cells. In addition, we analyzed the co-expression of two or more kinds of cytokines at the level of a single cell. The results showed that tonsillar CD4+ T cells exhibited polyfunctionality by co-expressing two to five kinds of cytokines in the same time. These data furnished a basic theory for further understanding the differentiation of polyfunctional Th cells in human tonsils and their functions in resisting invasive microorganisms.

Stromal interleukin-33 promotes regulatory T cell-mediated immunosuppression in head and neck squamous cell carcinoma and correlates with poor prognosis.

Regulatory T cells (Tregs) mediate immunosuppressive signals that can contribute to the progression of head and neck squamous cell carcinoma (HNSCC). Interleukin-33 (IL-33) is defined as an 'alarmin', an endogenous factor that is expressed during tissue and cell damage, which has been shown to promote Treg proliferation in non-lymphoid organs. However, the interaction between IL-33 and Tregs in the HNSCC tumor microenvironment remains uncertain. In this study, we examined IL-33+ and Foxp3+ cells by immunohistochemistry in 68 laryngeal squamous cell cancer patients, followed by functional analysis of IL-33 in Tregs. In addition, the suppressive function of Tregs was assessed by cell proliferation assays. The level of stromal IL-33 was significantly upregulated in advanced versus early stage HNSCC patients and positively correlated with Foxp3+ Treg infiltration as well as a poor prognosis. ST2 is regarded as the only receptor of IL-33. Infiltrated ST2-expressing Tregs were responsive to IL-33, and the percentage of Tregs was increased upon IL-33 stimulation. Functional investigation demonstrated that IL-33 increased the proportion of Foxp3+GATA3+ Tregs and improved the suppressive functions of Tregs by inducing IL-10 and TGF-ß1 as well as decreasing the proliferation of responder T cells. Blockade of ST2 abrogated the immunosuppression caused by IL-33. Our data demonstrate that stromal IL-33 both expands the Treg population and enhances their functions in the tumor microenvironment. Furthermore, stromal IL-33 has prognostic value for tumor progression. Thus, stromal IL-33 is a potential target for future HNSCC immunotherapy.

YAP Is Essential for Treg-Mediated Suppression of Antitumor Immunity.

Regulatory T cells (Treg) are critical for maintaining self-tolerance and immune homeostasis, but their suppressive function can impede effective antitumor immune responses. FOXP3 is a transcription factor expressed in Tregs that is required for their function. However, the pathways and microenvironmental cues governing FOXP3 expression and Treg function are not completely understood. Herein, we report that YAP, a coactivator of the Hippo pathway, is highly expressed in Tregs and bolsters FOXP3 expression and Treg function in vitro and in vivo. This potentiation stemmed from YAP-dependent upregulation of activin signaling, which amplifies TGFß/SMAD activation in Tregs. YAP deficiency resulted in dysfunctional Tregs unable to suppress antitumor immunity or promote tumor growth in mice. Chemical YAP antagonism and knockout or blockade of the YAP-regulated activin receptor similarly improved antitumor immunity. Thus, we identify YAP as an unexpected amplifier of a Treg-reinforcing pathway with significant potential as an anticancer immunotherapeutic target.Significance: Tregs suppress antitumor immunity, and pathways supporting their function can be novel immunotherapy targets. Here, the selective expression of YAP by Tregs, its importance for their function, and its unexpected enhancement of pro-Treg Activin/SMAD signaling are reported, as are validations of potential cancer-fighting antagonists of YAP and its regulatory targets. Cancer Discov; 8(8); 1026-43. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 899.

Excessive periostin expression and Th2 response in patients with nasal polyps: association with asthma.

BACKGROUND: Periostin has been shown to be upregulated in chronic rhinosinusitis with nasal polyps (CRSwNP), especially in the CRSwNP patients with asthma. However, the underlying mechanism that how periostin contributes to the polyp genesis remains unclear. METHODS: In this study, we collected 63 CRSwNP patients' nasal polyps (NPs) and 25 control subjects' uncinated tissues. The expressions of periostin, thymic stromal lymphopoietin (TSLP), and other proinflammatory cytokines were examined using IHC staining, qRT-PCR, Western blot (WB), ELISA and FACS. The eosinophil infiltration, phenotype profiles and clinical characteristics of 2 NP subtypes (eosinophilic and non-eosinophilic) were evaluated. We examined the effects and mechanisms of periostin on human nasal epithelial cells cultured at air-liquid interface (ALI). RESULTS: The expressions of periostin in NPs with asthma were higher than without asthma and the control nasal mucosa and positively associated with the TSLP (P<0.05). And the periostin levels was positively associated with the basement membrane thickness, goblet cell hyperplasia and tissue eosinophilia polyp tissues, as well as the clinical parameters (computed tomography scores, polyp size, and polyp recurrence after endoscopic surgery). In vitro experiments show that type 2 T-helper (Th2) cytokines interleukin-4 (IL-4), IL-13 and TGF-ß1 stimulates epithelial cells derived from polyp tissues to produce periostin through ERK and STAT6 signal pathways (P<0.05). Autocrine or recombinant periostin activates epithelial cells to produce TSLP via NF-κB signal pathways (P<0.05). The supernatant of periostin-treated epithelial cells activates dendritic cells (DCs), which subsequently induce naïve T cells to differentiate into Th2 cells and express IL-4 and IL-13. CONCLUSIONS: Our findings indicate periostin may play an important role in the polyp genesis, which can be considered as a therapeutic target for the management of CRSwNP.

MicroRNA-17 Modulates Regulatory T Cell Function by Targeting Co-regulators of the Foxp3 Transcription Factor.

Regulatory T (Treg) cells are important in maintaining self-tolerance and immune homeostasis. The Treg cell transcription factor Foxp3 works in concert with other co-regulatory molecules, including Eos, to determine the transcriptional signature and characteristic suppressive phenotype of Treg cells. Here, we report that the inflammatory cytokine interleukin-6 (IL-6) actively repressed Eos expression through microRNA-17 (miR-17). miR-17 expression increased in Treg cells in the presence of IL-6, and its expression negatively correlated with that of Eos. Treg cell suppressive activity was diminished upon overexpression of miR-17 in vitro and in vivo, which was mitigated upon co-expression of an Eos mutant lacking miR-17 target sites. Also, RNAi of miR-17 resulted in enhanced suppressive activity. Ectopic expression of miR-17 imparted effector-T-cell-like characteristics to Treg cells via the de-repression of genes encoding effector cytokines. Thus, miR-17 provides a potent layer of Treg cell control through targeting Eos and additional Foxp3 co-regulators.

Increased Expression of miR-146a in Children With Allergic Rhinitis After Allergen-Specific Immunotherapy.

PURPOSE: MicroRNAs (miRs) were recently recognized to be important for immune cell differentiation and immune regulation. However, whether miRs were involved in allergen-specific immunotherapy (SIT) remains largely unknown. This study sought to examine changes in miR-146a and T regulatory cells in children with persistent allergic rhinitis (AR) after 3 months of subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT). METHODS: Twenty-four HDM-sensitized children with persistent AR were enrolled and treated with SCIT (n=13) or SLIT (n=11) for 3 months. Relative miR-146a and Foxp3 mRNA expression, the TRAF6 protein level, and the ratio of post-treatment to baseline IL-10+CD4+ T cells between the SCIT and SLIT groups were examined in the peripheral blood mononuclear cells (PBMCs) of AR patients using quantitative reverse transcription polymerase chain reaction (qRT-PCR), flow cytometry, and Western blot analysis, respectively. Serum levels of IL-5 and IL-10 were determined using ELISA. RESULTS: After 3 months of SIT, both the TNSS and INSS scores were significantly decreased compared to the baseline value (P<0.01). The relative expression of miR-146a and Foxp3 mRNA was significantly increased after both SCIT and SLIT (P<0.01). The ratio of post-treatment to baseline IL-10⁺CD4⁺ T cells and the serum IL-10 level were significantly increased in both the SCIT and SLIT groups (P<0.01), whereas the TRAF6 protein level and serum IL-5 level were significantly decreased (P<0.01). No significant differences in these biomarkers were observed between the SCIT and SLIT groups. CONCLUSIONS: Our findings suggest that miR-146a and its related biomarkers may be comparably modulated after both SCIT and SLIT, highlighting miR-146a as a potential therapeutic target for the improved management of AR.

Expression of leukotriene and its receptors in eosinophilic chronic rhinosinusitis with nasal polyps.

BACKGROUND: Cysteinyl leukotriene (LT) has been proposed in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). This study sought to examine the expression of the LT receptor (LTR) in CRSwNP patients and evaluate the potential role of LTR antagonist (LTRA) in the management of eosinophilic CRSwNP (ECRS) patients. METHODS: Nasal polyps and uncinate process tissues were collected from 18 ECRS patients, 13 non-eosinophilic CRSwNP (non-ECRS) patients, and 16 control subjects. The messenger RNA (mRNA) and protein expression of LTR (cysteinyl leukotriene receptor 1 [CysLT1R] and cysteinyl leukotriene receptor 2 [CysLT2R]) was examined using quantitative reverse-transcription polymerase chain reaction (qRT-PCR), immunohistochemistry, and Western blot analysis. Moreover, the effects of LTRA and steroids on total nasal symptom scores (TNSS) of uncontrolled ECRS patients were evaluated. RESULTS: The mRNA and protein expression of CysLT1R and CysLT2R was significantly increased in polyp tissues compared with healthy controls (p < 0.05). Compared with the non-ECRS subset, the ECRS subset showed significantly increased expression of CysLT1R and CysLT2R, as well as leukotriene C4 (LTC4) and leukotriene D4 (LTC4) levels (p < 0.05). Moreover, combined LTRA and steroids significantly decreased TNSS more than steroids alone in uncontrolled ECRS patients (p < 0.01). CONCLUSION: Our findings indicate that LTR was differentially expressed between ECRS and non-ECRS patients, and that LTRA may be used as an additional therapy for ECRS patients.

Human IL-21+IFN-γ+CD4+ T cells in nasal polyps are regulated by IL-12.

In the previous study, we found that the levels of IL-21 in nasal polyps (NPs) were significantly increased and associated with polyp size and recurrence. However, it is unclear that the cell source of IL-21 and the regulation of IL-21 in NP tissues. In the present study, we isolated the lymphocytes from NP tissues, uncinate tissues and peripheral blood of patients with NPs. The cells were analyzed for cell surface markers, cytokines and transcriptional factors by flow cytometry. The results indicated that CD4(+) T cells were the major IL-21-expressing cells in NP tissues and the majority of IL-21 producing CD4(+) T cells co-expressed IFN-γ or IL-17A. IL-21(+)IFN-γ(+)CD4(+) T cells in NP tissues exhibited the features of both Tfh and Th1 cells which co-expressed significantly higher amount of CXCR5, ICOS, PD-1, Bcl-6 and T-bet than did IL-21(+)IFN-γ(-)CD4(+) T cells (p < 0.05). Treatment of the lymphocytes from NP tissues with IL-12 enhanced the production of IL-21 and IFN-γ, especially the frequency of IL-21(+)IFN(-)γ(+)CD4(+) T cells (p < 0.05). The blockade of IL-12 inhibited the production of IL-21 and IFN-γ (p < 0.05). These findings indicated that IL-12 positively enhanced the generation of IL-21(+)IFN-γ(+)CD4(+) T cells having the features of both Tfh and Th1 cells in NP tissues.