PPARγ inhibition promotes osteogenic differentiation of bone marrow mesenchymal stem cells and fracture healing.

This study aimed to explore the effects of peroxisome proliferator-activated receptor γ (PPARγ) inhibition on fracture healing of nonunion and the underlying mechanisms. Bone marrow mesenchymal stem cells (BMSCs) were treated with PPARγ antagonist GW9662 (5 µM, 10 µM). Alkaline phosphatase (ALP) staining and Alizarin Red S was used to assess early stage of osteogenesis and osteogenic differentiation. GW9662 (1 mg/kg/day) were administered intraperitoneally into the rats with bone fracture. Bone healing processes in the rat femur fracture model were recorded and assessed by radiographic methods on Weeks 8, 14, and 20 postoperation. Osteogenesis and angiogenesis at the fracture sites were evaluated by radiographic and histological methods on postoperative Week 20. GW9662 treatment increased ALP activity and Alp mRNA expression in rat BMSCs. Moreover, GW9662 administration increased matrix mineralization and mRNA and protein levels of Bmp2 and Runx2 in the BMSCs. In addition, GW9662 treatment improved radiographic score in the fracture rats and increased osteogenesis-related proteins, including type I collagen, osteopontin, and osteoglycin, in the bone tissues of the fracture sites. In conclusion, PPARγ inhibition promotes osteogenic differentiation of rat BMSCs, as well as improves the fracture healing of rats through Bmp2/Runx2 signaling pathway in the rat model of bone fracture.

Understanding metabolic alterations after SARS-CoV-2 infection: insights from the patients' oral microenvironmental metabolites.

BACKGROUND: Coronavirus disease 2019 is a type of acute infectious pneumonia and frequently confused with influenza since the initial symptoms. When the virus colonized the patient's mouth, it will cause changes of the oral microenvironment. However, few studies on the alterations of metabolism of the oral microenvironment affected by SARS-CoV-2 infection have been reported. In this study, we explored metabolic alterations of oral microenvironment after SARS-CoV-2 infection. METHODS: Untargeted metabolomics (UPLC-MS) was used to investigate the metabolic changes between oral secretion samples of 25 COVID-19 and 30 control participants. To obtain the specific metabolic changes of COVID-19, we selected 25 influenza patients to exclude the metabolic changes caused by the stress response of the immune system to the virus. Multivariate analysis (PCA and PLS-DA plots) and univariate analysis (students' t-test) were used to compare the differences between COVID-19 patients and the controls. Online hiplot tool was used to perform heatmap analysis. Metabolic pathway analysis was conducted by using the MetaboAnalyst 5.0 web application. RESULTS: PLS-DA plots showed significant separation of COVID-19 patients and the controls. A total of 45 differential metabolites between COVID-19 and control group were identified. Among them, 35 metabolites were defined as SARS-CoV-2 specific differential metabolites. Especially, the levels of cis-5,8,11,14,17-eicosapentaenoic acid and hexanoic acid changed dramatically based on the FC values. Pathway enrichment found the most significant pathways were tyrosine-related metabolism. Further, we found 10 differential metabolites caused by the virus indicating the body's metabolism changes after viral stimulation. Moreover, adenine and adenosine were defined as influenza virus-specific differential metabolites. CONCLUSIONS: This study revealed that 35 metabolites and tyrosine-related metabolism pathways were significantly changed after SARS-CoV-2 infection. The metabolic alterations of oral microenvironment in COVID-19 provided new insights into its molecular mechanisms for research and prognostic treatment.

Spectroscopy-Based Local Modeling Method for High-Throughput Quantification of Nucleic Acid Loading in Lipid Nanoparticles.

Lipid nanoparticles (LNPs) are the most widely investigated delivery systems for nucleic acid-based therapeutics and vaccines. Loading efficiency of nucleic acids may vary with formulation conditions, and it is considered one of the critical quality attributes of LNP products. Current analytical methods for quantification of cargo loading in LNPs often require external standard preparations and preseparation of unloaded nucleic acids from LNPs; therefore, they are subject to tedious and lengthy procedures, LNP stability, and unpredictable recovery rates of the separated analytes. Here, we developed a modeling approach, which was based on locally weighted regression (LWR) of ultraviolet (UV) spectra of unpurified samples, to quantify the loading of nucleic acid cargos in LNPs in-situ. We trained the model to automatically tune the training library space according to the spectral features of a query sample so as to robustly predict the nucleic acid cargo concentration and rank loading capacity with similar performance as the more complicated experimental approaches. Furthermore, we successfully applied the model to a wide range of nucleic acid cargo species, including antisense oligonucleotides, single-guided RNA, and messenger RNA, in varied lipid matrices. The LWR modeling approach significantly saved analytical time and efforts by facile UV scans of 96-well sample plates within a few minutes and with minimal sample preprocessing. Our proof-of-concept study presented the very first data mining and modeling strategy to quantify nucleic acid loading in LNPs and is expected to better serve high-throughput screening workflows, thereby facilitates early-stage optimization and development of LNP formulations.

Tie2-expressing monocytes as a novel angiogenesis-related cellular biomarker for non-small cell lung cancer.

To investigate the clinical value of Tie2-expressing monocytes (TEMs) in the early diagnosis of lung cancer and assess its correlation with angiogenesis, a total of 184 patients with non-small cell lung cancer (NSCLC), 101 patients with benign pulmonary disease (BPD), and 77 healthy controls were enrolled in our study. The distribution of TEMs in lung tissue was determined by immunofluorescence staining. Lung microvascular density was assessed by immunohistochemical staining. Receiver-operating characteristic (ROC) curve analysis was performed to assess the diagnostic value of TEM frequency. Patients with NSCLC were followed up for 26 months. We found that the TEM frequency in peripheral blood monocytes of patients with NSCLC was significantly greater than that in patients with BPD and healthy controls. TEM frequency showed a correlation with NSCLC recurrence. The majority of TEMs in tumor tissues were localized around blood vessels; tumoral TEM frequency showed a positive correlation with microvascular density. High percentage of TEMs in the peripheral blood was associated with poor overall survival. ROC curve analysis revealed the potential diagnostic value of circulating TEM frequency in NSCLC. Thus, we believe that TEM frequency is related to angiogenesis in tumor tissues and may serve as a diagnostic marker for NSCLC.

Seismic Slip-Pulse Experiments Simulate Induced Earthquake Rupture in the Groningen Gas Field.

Rock materials show dramatic dynamic weakening in large-displacement (m), high-velocity (∼1 m/s) friction experiments, providing a mechanism for the generation of large, natural earthquakes. However, whether such weakening occurs during induced M3-4 earthquakes (dm displacements) is unknown. We performed rotary-shear experiments on simulated fault gouges prepared from the source-, reservoir- and caprock formations present in the seismogenic Groningen gas field (Netherlands). Water-saturated gouges were subjected to a slip pulse reaching a peak circumferential velocity of 1.2-1.7 m/s and total displacements of 13-20 cm, at 2.5-20 MPa normal stress. The results show 22%-81% dynamic weakening within 5-12 cm of slip, depending on normal stress and gouge composition. At 20 MPa normal stress, dynamic weakening from peak friction coefficients of 0.4-0.9 to 0.19-0.27 was observed, probably through thermal pressurization. We infer that similar effects play a key role during induced seismic slip on faults in the Groningen and other reservoir systems.

ERRα negatively regulates type I interferon induction by inhibiting TBK1-IRF3 interaction.

Estrogen-related receptor α (ERRα) is a member of the nuclear receptor superfamily controlling energy homeostasis; however, its precise role in regulating antiviral innate immunity remains to be clarified. Here, we showed that ERRα deficiency conferred resistance to viral infection both in vivo and in vitro. Mechanistically, ERRα inhibited the production of type-I interferon (IFN-I) and the expression of multiple interferon-stimulated genes (ISGs). Furthermore, we found that viral infection induced TBK1-dependent ERRα stabilization, which in turn associated with TBK1 and IRF3 to impede the formation of TBK1-IRF3, IRF3 phosphorylation, IRF3 dimerization, and the DNA binding affinity of IRF3. The effect of ERRα on IFN-I production was independent of its transcriptional activity and PCG-1α. Notably, ERRα chemical inhibitor XCT790 has broad antiviral potency. This work not only identifies ERRα as a critical negative regulator of antiviral signaling, but also provides a potential target for future antiviral therapy.

Knockdown of lncRNA BDNF-AS suppresses neuronal cell apoptosis via downregulating miR-130b-5p target gene PRDM5 in acute spinal cord injury.

OBJECTIVE: The present study was designed to investigate the molecular mechanism and biological roles of lncRNA brain-derived neurotrophic factor antisense (lncRNA BDNF-AS) in acute spinal cord injury (ASCI). METHODS: The rat model of ASCI and hypoxic cellular model were established to detect the expression of BDNF-AS, miR-130b-5p, PR (PRDI-BF1 and RIZ) domain protein 5 (PRDM5) and cleaved caspase 3 (c-caspase 3) using qRT-PCR and western blot. Basso, Beattie and Bresnahan (BBB) score was carried out to assess neurological function. Flow cytometry was used to determine the apoptosis of neuronal cells. The association among BDNF-AS, miR-130b-5p and PRDM5 were disclosed by RNA immunoprecipitation (RIP) assay, RNA pull-down assay and dual-luciferase reporter assay. RESULTS: BDNF-AS, PRDM5 and c-caspase 3 expression were significantly upregulated, while miR-130b-5p was suppressed in the ASCI group and neuronal cells following hypoxia treatment. BDNF-AS knockdown inhibited neuronal cell apoptosis. Further studies indicated that BDNF-AS functioned as a competing endogenous RNA (ceRNA) by sponging miR-130b-5p in neuronal cells. Further investigations demonstrated that PRDM5 was a target of miR-130b-5p and BDNF-AS knockdown exerted anti-apoptotic effects via miR-130b-5p/PRDM5 axis. CONCLUSION: The lncRNA BDNF-AS/miR-130b-5p/PRDM5 axis might be a promising therapeutic target for ASCI.

Bacterial E3 Ubiquitin Ligase IpaH4.5 of Shigella flexneri Targets TBK1 To Dampen the Host Antibacterial Response.

IFN regulatory factors play a pivotal role in many cellular processes, including inflammatory and immune responses. Their activation is tightly regulated by TANK-binding kinase 1 (TBK1). In response to microbial components, TBK1 activates IFN regulatory factor 3 (IRF3) and cytokine expression. In this article, we show that TBK1 is a novel target of the IpaH4.5 protein, a Shigella type III effector possessing E3 ubiquitin ligase activity. Remarkably, IpaH4.5 interacts with TBK1 and promotes its K48-linked polyubiquitylation. Consequently, polyubiquitylated TBK1 undergoes proteasome-dependent degradation, which perturbs the phosphorylation, nuclear translocation, and activation of IRF3. Because IRF3 and TBK1 are required for restricting Shigella growth, we propose that the polyubiquitylation and degradation of TBK1 during Shigella infection are new bacterial strategies to modulate the host antibacterial responses.

Yersinia YopJ negatively regulates IRF3-mediated antibacterial response through disruption of STING-mediated cytosolic DNA signaling.

The Yersinia outer protein J (YopJ) plays a pivotal role in evading the host immune response and establishes a persistent infection in host cells after bacterial infection. YopJ is a cysteine protease and can act as a deubiquitinating enzyme that deubiquitinates several targets in multiple signaling pathways. Stimulator of interferon genes (STING) is a critical adapter for the induction of interferon regulatory factor 3 (IRF3) phosphorylation and subsequent production of the cytokines in response to nucleic acids in the cytoplasm. Our studies demonstrate that YopJ targets STING to inhibit IRF3 signaling. Specially, YopJ interacts with STING to block its ER-to-Golgi traffic and remove its K63-linked ubiquitination chains. Deubiquited STING perturbs the formation of STING-TBK1 complex and the activation of IRF3. The 172th cysteine of YopJ mediated STING deubiquitination and IRF3 signaling inhibition. Consequently, mice infected with WT and ΔYopJ/YopJ bacteria induced lower levels of IRF3 and IFN-ß, decreased inflammation and reduced staining of STING as compared to ΔYopJ and ΔYopJ/YopJ C172A strains infection. The data herein reveal a previously unrecognized mechanism by which YopJ modulates innate immune signaling.

MAVS Promotes Inflammasome Activation by Targeting ASC for K63-Linked Ubiquitination via the E3 Ligase TRAF3.

Stringent control of inflammasome signaling pathway is important for maintaining immunological balance, yet the molecular mechanisms responsible for its tight regulation are still poorly understood. In this study, we found that the signaling pathway dependent on mitochondrial antiviral signaling protein (MAVS) was required for the optimal activation of apoptosis-associated specklike protein (ASC)-dependent inflammasome. In particular, TNFR-associated factor 3 was found to be a direct E3 ligase for ASC. Ubiquitination of ASC at Lys(174) was critical for speck formation and inflammasome activation. Deficiency in MAVS or TNFR-associated factor 3 impaired ASC ubiquitination and cytosolic aggregates formation, resulting in reduced inflammasome response upon RNA virus infection. This study has identified a previously unrecognized role of MAVS in the regulation of inflammasome signaling and provided molecular insight into the mechanisms by which ubiquitination of ASC controls inflammasome activity through the formation of ASC specks.

Sleep disorders in patients with multiple sclerosis in China.

BACKGROUND: Poor sleep is common in patients with multiple sclerosis (MS). This study assessed the prevalence of poor sleep and investigated the potential impact factors that influence sleep quality of patients with MS. METHODS: A cross-sectional self-report survey of 231 patients with MS and 265 sex- and age-matched controls was conducted. Good sleepers and poor sleepers were separated by their global score on the Pittsburgh Sleep Quality Index (PSQI). Sociodemographic parameters, such as age, gender, and marital status, and clinical-demographic parameters, such as excessive daytime sleepiness (measured by the Epworth Sleepiness Scale), snoring, insomnia, obstructive sleep apnea, drugs, pain, depression, fatigue, and quality of life, were registered. Clinical and sociodemographic parameters were compared between patients with MS and controls and between good and poor sleepers among patients with MS. RESULTS: The prevalence of poor sleep in patients with MS was 64.9. Univariate analysis found that gender (p < 0.001), antidepressant drugs (p < 0.001), insomnia (p < 0.001), fatigue (p < 0.001), Epworth Sleepiness Scale (ESS) (p < 0.001), pain (p < 0.001), and depression (p < 0.001) were associated with sleep disorders. Multivariate analysis revealed that female gender, antidepressant drug treatment, and a high psychological burden of MS may be risk factors for poor sleep among patients with MS. CONCLUSIONS: Poor sleep is more common in patients with MS than in the general population. Sleep disorders should routinely be evaluated in patients with MS to improve the quality of sleep among them.

Elevated expression of TANK-binding kinase 1 enhances tamoxifen resistance in breast cancer.

Resistance to antiestrogens is one of the major challenges in breast cancer treatment. Although phosphorylation of estrogen receptor α (ERα) is an important factor in endocrine resistance, the contributions of specific kinases in endocrine resistance are still not fully understood. Here, we report that an important innate immune response kinase, the IκB kinase-related TANK-binding kinase 1 (TBK1), is a crucial determinant of resistance to tamoxifen therapies. We show that TBK1 increases ERα transcriptional activity through phosphorylation modification of ERα at the Ser-305 site. Ectopic TBK1 expression impairs the responsiveness of breast cancer cells to tamoxifen. By studying the specimens from patients with breast cancer, we find a strong positive correlation of TBK1 with ERα, ERα Ser-305, and cyclin D1. Notably, patients with tumors highly expressing TBK1 respond poorly to tamoxifen treatment and show high potential for relapse. Therefore, our findings suggest that TBK1 contributes to tamoxifen resistance in breast cancer via phosphorylation modification of ERα.

Sequential C-H Arylation and Enantioselective Hydrogenation Enables Ideal Asymmetric Entry to the Indenopiperidine Core of an 11ß-HSD-1 Inhibitor.

A concise asymmetric synthesis of an 11ß-HSD-1 inhibitor has been achieved using inexpensive starting materials with excellent step-economy at low catalyst loadings. The catalytic enantioselective total synthesis of 1 was accomplished in 7 steps and 38% overall yield aided by the development of an innovative, sequential strategy involving Pd-catalyzed pyridinium C-H arylation and Ir-catalyzed asymmetric hydrogenation of the resulting fused tricyclic indenopyridinium salt highlighted by the use of a unique P,N-ligand (MeO-BoQPhos) with 1000 ppm of [Ir(COD)Cl]2.

Ligand-Accelerated Stereoretentive Suzuki-Miyaura Coupling of Unprotected 3,3'-Dibromo-BINOL.

An efficient synthesis of the enantiomerically pure 3,3'-bis-arylated BINOL derivatives is accomplished through the palladium-catalyzed Suzuki-Miyaura coupling of the unprotected 3,3'-dibromo-BINOL with complete retention of enantiopurity. The active catalyst system Pd(OAc)2/BI-DIME has enabled mild reaction conditions at palladium loads as low as 500 ppm.

Downregulation of microRNA miR-526a by enterovirus inhibits RIG-I-dependent innate immune response.

UNLABELLED: Retinoic acid-inducible gene I (RIG-I) is an intracellular RNA virus sensor that induces type I interferon-mediated host-protective innate immunity against viral infection. Although cylindromatosis (CYLD) has been shown to negatively regulate innate antiviral response by removing K-63-linked polyubiquitin from RIG-I, the regulation of its expression and the underlying regulatory mechanisms are still incompletely understood. Here we show that RIG-I activity is regulated by inhibition of CYLD expression mediated by the microRNA miR-526a. We found that viral infection specifically upregulates miR-526a expression in macrophages via interferon regulatory factor (IRF)-dependent mechanisms. In turn, miR-526a positively regulates virus-triggered type I interferon (IFN-I) production, thus suppressing viral replication, the underlying mechanism of which is the enhancement of RIG-I K63-linked ubiquitination by miR-526a via suppression of the expression of CYLD. Remarkably, virus-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein, while ectopic miR-526a expression inhibits the replication of EV71 virus. The collective results of this study suggest a novel mechanism of the regulation of RIG-I activity during RNA virus infection by miR-526a and suggest a novel mechanism for the evasion of the innate immune response controlled by EV71. IMPORTANCE: RNA virus infection upregulates the expression of miR-526a in macrophages through IRF-dependent pathways. In turn, miR-526a positively regulates virus-triggered type I IFN production and inhibits viral replication, the underlying mechanism of which is the enhancement of RIG-I K-63 ubiquitination by miR-526a via suppression of the expression of CYLD. Remarkably, virus-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein; cells with overexpressed miR-526a were highly resistant to EV71 infection. The collective results of this study suggest a novel mechanism of the regulation of RIG-I activity during RNA virus infection by miR-526a and propose a novel mechanism for the evasion of the innate immune response controlled by EV71.

miR-526a regulates apoptotic cell growth in human carcinoma cells.

MicroRNAs (miRNAs) play vital roles in the regulation of cell cycle, cell growth, apoptosis, and tumorigenesis. Our previous studies showed that miR-526a positively regulated innate immune response by suppressing CYLD expression, however, the functional relevance of miR-526a expression and cell growth remains to be evaluated. In this study, miR-526a overexpression was found to promote cancer cell proliferation, migration, and anchor-independent colony formation. The molecular mechanism(s) of miR-526a-mediated growth stimulation is associated with rapid cell cycle progression and inhibition of cell apoptosis by targeting CYLD. Taken together, these results provide evidence to show the stimulatory role of miR-526a in tumor migration and invasion through modulation of the canonical NF-κB signaling pathway.

Development of an asymmetric synthesis of a chiral quaternary FLAP inhibitor.

A practical sequence involving a noncryogenic stereospecific boronate rearrangement followed by a robust formylation with an in situ generated DCM anion has been developed for the asymmetric construction of an all-carbon quaternary stereogenic center of a FLAP inhibitor. The key boronate rearrangement was rendered noncryogenic and robust by using LDA as the base and instituting an in situ trapping of the unstable lithiated benzylic carbamate with the boronic ester. A similar strategy was implemented for the DCM formylation reaction. It was found that the 1,2-boronate rearrangement for the formylation reaction could be temperature-controlled, thus preventing overaddition of the DCM anion and rendering the process reproducible. The robust stereospecific boronate rearrangement and formylation were utilized for the practical asymmetric synthesis of a chiral quaternary FLAP inhibitor.

An epidemiologic study of restless legs syndrome among Chinese children and adolescents.

To determine the prevalence of restless legs syndrome (RLS) in Chinese children and adolescents as well as the impact of the disorder on 8-11 and 12-17 years old. This population-based study was conducted in five primary schools and seven high schools, which were randomly selected in Henan province, China. A total of 6792 students aged 8-17 years old were given a questionnaire that included the adult diagnostic criteria of RLS proposed by the International Restless Legs Study Group. Subjects who answered "yes" to all four questions were selected for a face-to-face interview to confirm RLS diagnosis. Individuals with definite RLS were then administered another questionnaire to survey RLS symptoms and perceived consequences. The prevalence of definite RLS in Chinese children and adolescents was 2.2 % (141/6437), with a prevalence of 1.8 % in the 8-11 years old age group and 2.4 % in the 12-17 years old age group. RLS was found to be more prevalent in females (2.7 %) than in males (1.7 %) (P = 0.008), and the prevalence of RLS was determined to increase with age. Sleep disturbance was the most common symptom of RLS in children and adolescents. Various consequences were attributed to RLS, with participants reporting that they dreaded the arrival of evening/night most frequently, followed by the description that RLS had a negative impact on mood. These data suggest that RLS is prevalent in Chinese children and adolescents, and that those affected by this disorder suffer from disruptions to sleep and daytime function.

Restless legs syndrome and hypertension in Chinese pregnant women.

Hypertension is a common complication of pregnancy, and studies show that pregnant women are more likely to suffer from restless legs syndrome (RLS). Pregnant women with hypertension and RLS often experience disrupted sleep patterns because of activation of the nervous system. The present study aimed to clarify the relationship between hypertension and RLS in pregnant women, and their impact on sleep. We enrolled 3,781 pregnant women who were admitted at our hospital for delivery between May 2011 and May 2014. The face-to-face questionnaire used to gather data included the International RLS Study Group criteria for diagnosis, Pittsburgh Sleep Quality Index (PSQI), Epworth Sleepiness Scale (ESS), and hypertension diagnosis. Depending on the time of occurrence of hypertension, it was divided into two different types: pregnancy-induced hypertension and chronic hypertension in pregnancy. Out of 3,781 patients, 453 fulfilled the diagnostic criteria for RLS and 486 met the diagnostic criteria for hypertension. Among patients with RLS, prophylactic iron supplementation was less frequently taken during pregnancy. Pregnancy-induced hypertension, rather than chronic hypertension in pregnancy, was found to be more frequent in patients with RLS; pregnant women with RLS had higher PSQI and ESS scores than pregnant controls. In our study, RLS was frequent in pregnant women, especially in those without prophylactic iron supplementation. Patients with RLS described more serious sleep disruption and excessive daytime sleepiness (EDS). In addition, pregnancy-induced hypertension was more common in patients with RLS.

Restless legs syndrome among pregnant women in China: prevalence and risk factors.

BACKGROUND: The goal of this study was to identify the prevalence and clinical correlates and severity of restless legs syndrome (RLS) among pregnant women in mainland China. METHODS: This cross-sectional study enrolled 1584 women (18-40 years old) who came to a prenatal outpatient clinic to consult an obstetrician. Pregnant women were studied in each trimester, and assessments included interviews about RLS symptoms and related questions. Standardized questionnaires include the International Restless Syndrome Scale and the Pittsburgh Sleep Quality Questionnaire. Blood tests included levels of hemoglobin and mean corpuscular volume. RESULTS: RLS was diagnosed in 177 of 1584 women (11.2%); 4.2% were categorized as having pre-existing RLS and 54.8% reported onset of RLS symptoms after the 24th week. Multivariate analysis revealed that anemia was positively correlated with RLS. For the participants who first experienced RLS in pregnancy, RLS severity in the third trimester was more severe when compared with the first and second trimesters. Sleep disorders occurred more frequently in the third trimester. CONCLUSIONS: In our study, RLS was frequent in pregnant Chinese women, and anemia was identified as an independent predictor of the disease. Further, most participants reported their symptoms during the third trimester, and the severity of RLS and sleep disorders of participants was more prominent in the third trimester.