Transport and inhibition mechanisms of the human noradrenaline transporter.

The noradrenaline transporter (also known as norepinephrine transporter) (NET) has a critical role in terminating noradrenergic transmission by utilizing sodium and chloride gradients to drive the reuptake of noradrenaline (also known as norepinephrine) into presynaptic neurons1-3. It is a pharmacological target for various antidepressants and analgesic drugs4,5. Despite decades of research, its structure and the molecular mechanisms underpinning noradrenaline transport, coupling to ion gradients and non-competitive inhibition remain unknown. Here we present high-resolution complex structures of NET in two fundamental conformations: in the apo state, and bound to the substrate noradrenaline, an analogue of the χ-conotoxin MrlA (χ-MrlAEM), bupropion or ziprasidone. The noradrenaline-bound structure clearly demonstrates the binding modes of noradrenaline. The coordination of Na+ and Cl- undergoes notable alterations during conformational changes. Analysis of the structure of NET bound to χ-MrlAEM provides insight into how conotoxin binds allosterically and inhibits NET. Additionally, bupropion and ziprasidone stabilize NET in its inward-facing state, but they have distinct binding pockets. These structures define the mechanisms governing neurotransmitter transport and non-competitive inhibition in NET, providing a blueprint for future drug design.

Hydrophobic tagging of small molecules: an overview of the literature and future outlook.

INTRODUCTION: Hydrophobic tagging (HyT) technology presents a distinct therapeutic strategy diverging from conventional small molecule drugs, providing an innovative approach to drug design. This review aims to provide an overview of the HyT literature and future outlook to offer guidance for drug design. AREAS COVERED: In this review, the authors introduce the composition, mechanisms and advantages of HyT technology, as well as summarize the detailed applications of HyT technology in anti-cancer, neurodegenerative diseases (NDs), autoimmune disorders, cardiovascular diseases (CVDs), and other fields. Furthermore, this review discusses key aspects of the future development of HyT molecules. EXPERT OPINION: HyT emerges as a highly promising targeted protein degradation (TPD) strategy, following the successful development of proteolysis targeting chimeras (PROTAC) and molecular glue. Based on exploring new avenues, modification of the HyT molecule itself potentially enhances the technology. Improved synthetic pathways and emphasis on pharmacokinetic (PK) properties will facilitate the development of HyT. Furthermore, elucidating the biochemical basis by which the compound's hydrophobic moiety recruits the protein homeostasis network will enable the development of more precise assays that can guide the optimization of the linker and hydrophobic moiety.

Discovery of a Novel Thienopyrimidine Compound as a Urate Transporter 1 and Glucose Transporter 9 Dual Inhibitor with Improved Efficacy and Favorable Druggability.

Gout and hyperuricemia are metabolic diseases characterized with high serum uric acid (SUA) levels that significantly impact human health. Lesinurad, a uricosuric agent, is limited to concurrent use with xanthine oxidase inhibitors (XOIs) in clinical practice due to its restricted efficacy and potential nephrotoxicity. Herein, extensive structural modifications of lesinurad were conducted through scaffold hopping and substituent modification strategies, affording 54 novel derivatives containing pyrimidine-fused cyclic structures. Notably, the thienopyrimidine compound 29 demonstrated a remarkable 2-fold increase in SUA-lowering in vivo activity compared to lesinurad, while exhibiting potent inhibitory activity against the urate transporter 1 (URAT1, IC50 = 2.01 µM) and glucose transporter 9 (GLUT9, IC50 = 18.21 µM). Furthermore, it possessed a lower effective dosage of 0.5 mg/kg, favorable safety profile without any apparent acute toxicity at doses of 1000 mg/kg, and improved pharmacokinetic properties. Overall, we have discovered an efficacious URAT1/GLUT9 dual inhibitor for inhibiting urate reabsorption with favorable pharmacokinetic profiles.

[Therapeutic effect of ursodeoxycholic acid-berberine supramolecular nanoparticles on ulcerative colitis based on supramolecular system induced by weak bond].

Ulcerative colitis(UC) is a recurrent, intractable inflammatory bowel disease. Coptidis Rhizoma and Bovis Calculus, serving as heat-clearing and toxin-removing drugs, have long been used in the treatment of UC. Berberine(BBR) and ursodeoxycholic acid(UDCA), the main active components of Coptidis Rhizoma and Bovis Calculus, respectively, were employed to obtain UDCA-BBR supramolecular nanoparticles by stimulated co-decocting process for enhancing the therapeutic effect on UC. As revealed by the characterization of supramolecular nanoparticles by field emission scanning electron microscopy(FE-SEM) and dynamic light scattering(DLS), the supramolecular nanoparticles were tetrahedral nanoparticles with an average particle size of 180 nm. The molecular structure was described by ultraviolet spectroscopy, fluorescence spectroscopy, infrared spectroscopy, high-resolution mass spectrometry, and hydrogen-nuclear magnetic resonance(H-NMR) spectroscopy. The results showed that the formation of the supramolecular nano-particle was attributed to the mutual electrostatic attraction and hydrophobic interaction between BBR and UDCA. Additionally, supramolecular nanoparticles were also characterized by sustained release and pH sensitivity. The acute UC model was induced by dextran sulfate sodium(DSS) in mice. It was found that supramolecular nanoparticles could effectively improve body mass reduction and colon shortening in mice with UC(P<0.001) and decrease disease activity index(DAI)(P<0.01). There were statistically significant differences between the supramolecular nanoparticles group and the mechanical mixture group(P<0.001, P<0.05). Enzyme-linked immunosorbent assay(ELISA) was used to detect the serum levels of tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6), and the results showed that supramolecular nanoparticles could reduce serum TNF-α and IL-6 levels(P<0.001) and exhibited an obvious difference with the mechanical mixture group(P<0.01, P<0.05). Flow cytometry indicated that supramolecular nanoparticles could reduce the recruitment of neutrophils in the lamina propria of the colon(P<0.05), which was significantly different from the mechanical mixture group(P<0.05). These findings suggested that as compared with the mechanical mixture, the supramolecular nanoparticles could effectively improve the symptoms of acute UC in mice. The study provides a new research idea for the poor absorption of small molecules and the unsatisfactory therapeutic effect of traditional Chinese medicine and lays a foundation for the research on the nano-drug delivery system of traditional Chinese medicine.

Targeting hepatitis B virus cccDNA levels: Recent progress in seeking small molecule drug candidates.

Hepatitis B virus (HBV) infection is a major global health problem that puts people at high risk of death from cirrhosis and liver cancer. The presence of covalently closed circular DNA (cccDNA) in infected cells is considered to be the main obstacle to curing chronic hepatitis B. At present, the cccDNA cannot be completely eliminated by standard treatments. There is an urgent need to develop drugs or therapies that can reduce HBV cccDNA levels in infected cells. We summarize the discovery and optimization of small molecules that target cccDNA synthesis and degradation. These compounds are cccDNA synthesis inhibitors, cccDNA reducers, core protein allosteric modulators, ribonuclease H inhibitors, cccDNA transcriptional modulators, HBx inhibitors and other small molecules that reduce cccDNA levels.

A case report and literature review of Carney complex with atrial adenomyxoma.

BACKGROUND: Carney complex (CNC) is a rare multiple endocrine neoplasia syndrome characterized by mucocutaneous lentigines/ blue nevi, cardiac myxoma and endocrine overactivity. Here, we report a CNC case with PRKAR1A gene mutation characterized by left atrial adenomyxoma to explore the diagnosis and treatment of CNC. CASE PRESENTATION: A 42-year-old woman with a history of cardiac tumour surgery presented with typical features of Cushing syndrome, including central obesity, buffalo hump, mild facial plethora, purple striae on the lower abdomen, and spotty skin pigmentation. Left atrial adenomyxoma and thyroid papillary carcinoma were identified by postoperative histologic assays. Genetic screening revealed a pathogenic germline heterozygous mutation of c.682C > T (p.R228X) in exon 7 of the PRKAR1A gene. The clinical features and normal ACTH levels suggest this patient suffered the ACTH-independent primary pigmented nodular adrenocortical disease (PPNAD) with cyclic hypercortisolism or ACTH-dependent Cushing syndrome. CONCLUSION: CNC is uncommon, however, if a patient develops clinical features involving multiple endocrine and non-endocrine tumors, especially Cushing syndrome and cardiac myxoma, CNC should be considered. Genetic analysis is recommended in patients with suspected CNC.

Medicinal chemistry insights into antiviral peptidomimetics.

The (re)emergence of multidrug-resistant viruses and the emergence of new viruses highlight the urgent and ongoing need for new antiviral agents. The use of peptidomimetics as therapeutic drugs has often been associated with advantages, such as enhanced binding affinity, improved metabolic stability, and good bioavailability profiles. The development of novel antivirals is currently driven by strategies of converting peptides into peptidomimetic derivatives. In this review, we outline different structural modification design strategies for developing novel peptidomimetics as antivirals, involving N- or C-cap terminal structure modifications, pseudopeptides, amino acid modifications, inverse-peptides, cyclization, and molecular hybridization. We also present successful recent examples of peptidomimetic designs.

Discovery and Mechanistic Investigation of Piperazinone Phenylalanine Derivatives with Terminal Indole or Benzene Ring as Novel HIV-1 Capsid Modulators.

HIV-1 capsid (CA) performs multiple roles in the viral life cycle and is a promising target for antiviral development. In this work, we describe the design, synthesis, assessment of antiviral activity, and mechanistic investigation of 20 piperazinone phenylalanine derivatives with a terminal indole or benzene ring. Among them, F2-7f exhibited moderate anti-HIV-1 activity with an EC50 value of 5.89 µM, which was slightly weaker than the lead compound PF74 (EC50 = 0.75 µM). Interestingly, several compounds showed a preference for HIV-2 inhibitory activity, represented by 7f with an HIV-2 EC50 value of 4.52 µM and nearly 5-fold increased potency over anti-HIV-1 (EC50 = 21.81 µM), equivalent to PF74 (EC50 = 4.16 µM). Furthermore, F2-7f preferred to bind to the CA hexamer rather than to the monomer, similar to PF74, according to surface plasmon resonance results. Molecular dynamics simulation indicated that F2-7f and PF74 bound at the same site. Additionally, we computationally analyzed the ADMET properties for 7f and F2-7f. Based on this analysis, 7f and F2-7f were predicted to have improved drug-like properties and metabolic stability over PF74, and no toxicities were predicted based on the chemotype of 7f and F2-7f. Finally, the experimental metabolic stability results of F2-7f in human liver microsomes and human plasma moderately correlated with our computational prediction. Our findings show that F2-7f is a promising small molecule targeting the HIV-1 CA protein with considerable development potential.

Discovery and Crystallographic Studies of Trisubstituted Piperazine Derivatives as Non-Covalent SARS-CoV-2 Main Protease Inhibitors with High Target Specificity and Low Toxicity.

The continuous spread of SARS-CoV-2 calls for more direct-acting antiviral agents to combat the highly infectious variants. The main protease (Mpro) is an promising target for anti-SARS-CoV-2 drug design. Here, we report the discovery of potent non-covalent non-peptide Mpro inhibitors featuring a 1,2,4-trisubstituted piperazine scaffold. We systematically modified the non-covalent hit MCULE-5948770040 by structure-based rational design combined with multi-site binding and privileged structure assembly strategies. The optimized compound GC-14 inhibits Mpro with high potency (IC50 = 0.40 µM) and displays excellent antiviral activity (EC50 = 1.1 µM), being more potent than Remdesivir. Notably, GC-14 exhibits low cytotoxicity (CC50 > 100 µM) and excellent target selectivity for SARS-CoV-2 Mpro (IC50 > 50 µM for cathepsins B, F, K, L, and caspase 3). X-ray co-crystal structures prove that the inhibitors occupy multiple subpockets by critical non-covalent interactions. These studies may provide a basis for developing a more efficient and safer therapy for COVID-19.

Novel urate transporter 1 (URAT1) inhibitors: a review of recent patent literature (2020-present).

INTRODUCTION: The urate transporter 1 (URAT1) is a membrane transporter located in the apical membrane of human renal proximal tubule epithelial cells, which mediates most of the reabsorption of urate. Hyperuricemia (HUA) is a common disease caused by metabolic disorders, which has been considered as the key factor of gout. Approximately 90% of patients suffer from hyperuricemia due to insufficient or poor uric acid excretion. Therefore, the drug design of URAT1 inhibitors targeting improve the renal urate excretion by reducing the reabsorption of urate anions represent a hot topic in searching for anti-gout drugs currently. AREAS COVERED: In this review, we summarize URAT1 inhibitors patents reported since 2020 to present through the public database at https://worldwide.espacenet.com and some medicinal chemistry strategies employed to develop novel drug candidates. EXPERT OPINION: Ligand-based drug design (LBDD) strategies have been frequently used developing new URAT1 inhibitors. Meanwhile, the discovery of dual drugs targeting both inhibition of xanthine oxidase (XOD) and URAT1 may be an emerging horizon for designing novel uric acid-lowering candidates in future. Furthermore, advanced techniques in the field of molecular biology and computer science can increase the chances to discover and/or optimize URAT1 inhibitors, contributing to the development of novel drug candidates.

Inverse correlation between serum irisin and cardiovascular risk factors among Chinese overweight/obese population.

BACKGROUND: Irisin is a novel myokine associated with obesity, which is a traditional cardiovascular risk factor (CVRF). The present study aimed to investigate the association between serum irisin and a single CVRF as well as the clustering of CVRFs among Chinese overweight/obese population. METHODS: A total of 98 overweight and 93 obese subjects without clinical treatments were enrolled in this study. Subjects were then divided into two groups, based on the serum irisin level: a low irisin group (1.10-13.44 ng/ml) and a high irisin group (13.49-29.9 ng/ml). The clustering of CVRFs, smoking, diabetes mellitus, dyslipidemia and hypertension, was classified as 0, 1, 2 and ≥ 3 CVRFs. The demographic and baseline clinical characteristics of all participants were collected and serum irisin was measured. RESULTS: The high serum irisin group had significantly higher high-density lipoprotein cholesterol but lower fasting plasma glucose than the low serum irisin group. Additionally, the high serum irisin group had a significantly lower prevalence of smoking, diabetes mellitus and dyslipidemia than the low serum irisin group. Increased serum irisin was significantly associated with a reduced risk of smoking and dyslipidemia in both the unadjusted and adjusted models. Furthermore, high serum irisin significantly reduced the risk of the prevalence of 1, 2 and ≥ 3 CVRFs. CONCLUSIONS: among the Chinese overweight/obese populations, high serum irisin is negatively associated with smoking, dyslipidemia and the clustering of CVRFs. Thus, high serum irisin is potentially associated with a low risk of cardiovascular diseases in the Chinese overweight/obese population.

Discovery of potent and selective Cdc25 phosphatase inhibitors via rapid assembly and in situ screening of Quinonoid-focused libraries.

Cell division cycle 25 (Cdc25) phosphatase is an attractive target for drug discovery. The rapid assembly and in situ screening of focused combinatorial fragment libraries using efficient modular reactions is a highly robust strategy for discovering bioactive molecules. In this study, we have utilized miniaturized synthesis to generate several quinonoid-focused libraries, by standard CuAAC reaction and HBTU-based amide coupling chemistry. Then the enzyme inhibition screening afforded some potent and selective Cdc25s inhibitors. Compound M5N36 (Cdc25A: IC50 = 0.15 ± 0.05 µM; Cdc25B: IC50 = 0.19 ± 0.06 µM; Cdc25C: IC50 = 0.06 ± 0.04 µM) exhibited higher inhibitory activity than the initial lead NSC663284 (Cdc25A: IC50 = 0.27 ± 0.02 µM; Cdc25B: IC50 = 0.42 ± 0.01 µM; Cdc25C: IC50 = 0.23 ± 0.01 µM). Moreover, M5N36 displayed about three-fold more potent against Cdc25C than Cdc25A and B, indicating that M5N36 could act as a relatively selective Cdc25C inhibitor. Cell viability evaluation, western blotting and molecular simulations provided a mechanistic understanding of the activity of M5N36. It showed promising anti-growth activity against the MDA-MB-231 cell line and desirable predicted physicochemical properties. Overall, M5N36 was proven to be a promising novel Cdc25C inhibitor.

Recent advances of quinones as a privileged structure in drug discovery.

Privileged structures are conductive to discover novel bioactive substances because they can bind to multiple targets with high affinity. Quinones are considered to be a privileged structure and useful template for the design of new compounds with potential pharmacological activity. This article presents the recent developments (2014-2021 update) of quinones in the fields of antitumor, antibacterial, antifungal, antiviral, anti-Alzheimer's disease (AD) and antimalarial, mainly focusing on biological activities, structural modification and mechanism of action.

A Hierarchical Approach Using Marginal Summary Statistics for Multiple Intermediates in a Mendelian Randomization or Transcriptome Analysis.

Previous research has demonstrated the usefulness of hierarchical modeling for incorporating a flexible array of prior information in genetic association studies. When this prior information consists of estimates from association analyses of single-nucleotide polymorphisms (SNP)-intermediate or SNP-gene expression, a hierarchical model is equivalent to a 2-stage instrumental or transcriptome-wide association study (TWAS) analysis, respectively. We propose to extend our previous approach for the joint analysis of marginal summary statistics to incorporate prior information via a hierarchical model (hJAM). In this framework, the use of appropriate estimates as prior information yields an analysis similar to Mendelian randomization (MR) and TWAS approaches. hJAM is applicable to multiple correlated SNPs and intermediates to yield conditional estimates for the intermediates on the outcome, thus providing advantages over alternative approaches. We investigated the performance of hJAM in comparison with existing MR and TWAS approaches and demonstrated that hJAM yields an unbiased estimate, maintains correct type-I error, and has increased power across extensive simulations. We applied hJAM to 2 examples: estimating the causal effects of body mass index (GIANT Consortium) and type 2 diabetes (DIAGRAM data set, GERA Cohort, and UK Biobank) on myocardial infarction (UK Biobank) and estimating the causal effects of the expressions of the genes for nuclear casein kinase and cyclin dependent kinase substrate 1 and peptidase M20 domain containing 1 on the risk of prostate cancer (PRACTICAL and GTEx).

Brachial-ankle pulse wave velocity is associated with the risk of osteoporosis: a cross-sectional evidence from a Chinese community-based cohort.

BACKGROUND: Association of arterial stiffness and osteoporosis has been well documented in elderly population. However, it is not clear whether they co-progress from the early stages through common mechanisms. The object of this study was to evaluate possible associations between arterial stiffness and osteoporosis by measuring brachial-ankle pulse wave velocity (baPWV) and the Osteoporosis Self-Assessment Tool for Asia (OSTA) index among a healthy population of Chinese aged 40 years and older. Whether baPWV can be used as a predictor of osteoporosis on OSTA was further assessed. METHODS: This study was cross-sectional in design. Of 3984 adults aged 40 years and older in the Yunyan district of Guiyang (Guizhou, China) who underwent both OSTA and baPWV measurements within 1 month, 1407 were deemed eligible for inclusion (women, 1088; men, 319). RESULTS: The mean baPWV was 1475 ± 302 cm/s (range,766-3459 cm/s). baPWV in 110 individuals with high risk of osteoporosis (OSTA index < - 4) was higher than that of individuals with non-high risk (1733 ± 461 cm/s vs. 1447 ± 304 cm/s, P < 0.001). OSTA index was negatively correlated with baPWV(ρ = - 0.296, P < 0.001) after adjusting for age, sex, body mass index, waist circumference, diastolic blood pressure, and creatinine clearance rate. baPWV was an independent predictor for the presence of high risk of osteoporosis (ß = - 0.001, P < 0.001) and was consistent across age and sex subgroups, and the optimal baPWV cutoff value for predicting the presence of high risk of osteoporosis and fracture was 1693 cm/s. The AUC was 0.722 (95% confidence interval [CI], 0.667-0.777; P < 0.001, sensitivity of 52.8% and specificity of 83.6%). CONCLUSIONS: We conclude that arterial stiffness measured by baPWV is well correlated with the severity of osteoporosis evaluated by OSTA. baPWV index may be a valuable tool for identifying individuals with risk of developing osteoporosis.

Irisin plays an important role in the outcomes of newly diagnosed prediabetes in adults in Guiyang, China.

AIMS/INTRODUCTION: To explore the potential role of irisin in the outcomes of newly diagnosed prediabetes. MATERIALS AND METHODS: Data were obtained from the Guiyang subcenter of the Risk Evaluation of cAncers in Chinese diabeTic Individuals: a lONgitudinal (REACTION) study. A total of 2,530 participants had newly diagnosed prediabetes at baseline and completed follow up. The nested 1:1 case-control study included 161 participants who developed diabetes mellitus at follow up, and 161 age- and sex-matched controls. The follow-up study included 86 matched case-control pairs. Fasting serum irisin levels were measured using enzyme-linked immunosorbent assay. RESULTS: Baseline serum irisin levels were higher in the cases than in the controls (P = 0.002); high baseline serum irisin levels were an independent risk factor for the development of diabetes (odds ratio 1.235, 95% confidence interval 1.025-1.488). After adjustment for age, sex, body mass index, glycated hemoglobin (HbA1c), smoking, exercise, and family history of diabetes, subjects in the highest quartile of irisin levels had a higher risk of diabetes than those in the lowest quartile (odds ratio 3.065, 95% confidence interval 1.511-6.218). The extent of decrease in irisin levels during follow-up was greater in the cases than in the controls (P < 0.001). Baseline serum irisin levels were positively correlated with the extent of decrease in irisin during follow-up (r = 0.773, P < 0.001). After adjustment for confounding factors, subjects with a decrease of irisin above the median had much higher risk for diabetes (odds ratio 5.077, 95% confidence interval 2.112-12.206). CONCLUSIONS: Irisin might play an important role in the outcomes of newly diagnosed prediabetes in adults in Guiyang, and can predict the risk for developing diabetes in these individuals.

Design, Synthesis, and Mechanism Study of Benzenesulfonamide-Containing Phenylalanine Derivatives as Novel HIV-1 Capsid Inhibitors with Improved Antiviral Activities.

The HIV-1 CA protein has gained remarkable attention as a promising therapeutic target for the development of new antivirals, due to its pivotal roles in HIV-1 replication (structural and regulatory). Herein, we report the design and synthesis of three series of benzenesulfonamide-containing phenylalanine derivatives obtained by further structural modifications of PF-74 to aid in the discovery of more potent and drug-like HIV-1 CA inhibitors. Structure-activity relationship studies of these compounds led to the identification of new phenylalanine derivatives with a piperazinone moiety, represented by compound 11l, which exhibited anti-HIV-1NL4-3 activity 5.78-fold better than PF-74. Interestingly, 11l also showed anti-HIV-2ROD activity (EC50 = 31 nM), with almost 120 times increased potency over PF-74. However, due to the higher significance of HIV-1 as compared to HIV-2 for the human population, this manuscript focuses on the mechanism of action of our compounds in the context of HIV-1. SPR studies on representative compounds confirmed CA as the binding target. The action stage determination assay demonstrated that these inhibitors exhibited antiviral activities with a dual-stage inhibition profile. The early-stage inhibitory activity of compound 11l was 6.25 times more potent as compared to PF-74 but appeared to work via the accelerating capsid core assembly rather than stabilization. However, the mechanism by which they exert their antiviral activity in the late stage appears to be the same as PF-74 with less infectious HIV-1 virions produced in their presence, as judged p24 content studies. MD simulations provided the key rationale for the promising antiviral potency of 11l. Additionally, 11l exhibited a modest increase in HLM and human plasma metabolic stabilities as compared to PF-74, as well as a moderately improved pharmacokinetic profile, favorable oral bioavailability, and no acute toxicity. These studies provide insights and serve as a starting point for subsequent medicinal chemistry efforts in optimizing these promising HIV inhibitors.

Impact of different fixed flow sampling protocols on flow-independent exhaled nitric oxide parameter estimates using the Bayesian dynamic two-compartment model.

Exhaled nitric oxide (FeNO) is an established respiratory biomarker with clinical applications in the diagnosis and management of asthma. Because FeNO depends strongly on the flow (exhalation) rate, early protocols specified that measurements should be taken when subjects exhaled at a fixed rate of 50 ml/s. Subsequently, multiple flow (or "extended") protocols were introduced which measure FeNO across a range of fixed flow rates, allowing estimation of parameters including Caw NO and CA NO which partition the physiological sources of NO into proximal airway wall tissue and distal alveolar regions (respectively). A recently developed dynamic model of FeNO uses flow-concentration data from the entire exhalation maneuver rather than plateau means, permitting estimation of Caw NO and CA NO from a wide variety of protocols. In this paper, we use a simulation study to compare Caw NO and CA NO estimation from a variety of fixed flow protocols, including: single maneuvers (30, 50,100, or 300 ml/s) and three established multiple maneuver protocols. We quantify the improved precision with multiple maneuvers and the importance of low flow maneuvers in estimating Caw NO. We conclude by applying the dynamic model to FeNO data from 100 participants of the Southern California Children's Health Study, establishing the feasibility of using the dynamic method to reanalyze archived online FeNO data and extract new information on Caw NO and CA NO in situations where these estimates would have been impossible to obtain using traditional steady-state two compartment model estimation methods.

Perfluoroalkyl substances and severity of nonalcoholic fatty liver in Children: An untargeted metabolomics approach.

BACKGROUND: Toxicant-associated steatohepatitis has been described in adults but less is known regarding the role of toxicants in liver disease of children. Perfluoroalkyl substances (PFAS) cause hepatic steatosis in rodents, but few previous studies have examined PFAS effects on severity of liver injury in children. OBJECTIVES: We aimed to examine the relationship of PFAS to histologic severity of nonalcoholic fatty liver disease (NAFLD) in children. METHODS: Seventy-four children with physician-diagnosed NAFLD were recruited from Children's Healthcare of Atlanta between 2007 and 2015. Biopsy-based liver histological features were scored for steatosis, lobular and portal inflammation, ballooning, and fibrosis. Plasma concentrations of perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS) and perfluorohexane sulfonic acid (PFHxS), and untargeted plasma metabolomic profiling, were determined using liquid chromatography with high-resolution mass spectrometry. A metabolome-wide association study coupled with pathway enrichment analysis was performed to evaluate metabolic dysregulation associated with PFAS. A structural integrated analysis was applied to identify latent clusters of children with more severe form of NAFLD based on their PFAS levels and metabolite pattern. RESULTS: Patients were 7-19 years old, mostly boys (71%), Hispanic (51%), and obese (85%). The odds of having nonalcoholic steatohepatitis (NASH), compared to children with steatosis alone, was significantly increased with each interquartile range (IQR) increase of PFOS (OR: 3.32, 95% CI: 1.40-7.87) and PFHxS (OR: 4.18, 95% CI: 1.64-10.7). Each IQR increase of PFHxS was associated with increased odds for liver fibrosis (OR: 4.44, 95% CI: 1.34-14.8), lobular inflammation (OR: 2.87, 95% CI: 1.12-7.31), and higher NAFLD activity score (ß coefficient 0.46; 95% CI: 0.03, 0.89). A novel integrative analysis identified a cluster of children with NASH, characterized by increased PFAS levels and altered metabolite patterns including higher plasma levels of phosphoethanolamine, tyrosine, phenylalanine, aspartate and creatine, and decreased plasma levels of betaine. CONCLUSIONS: Ηigher PFAS exposure was associated with more severe disease in children with NAFLD. PFAS may be an important toxicant contributing to NAFLD progression; however larger, longitudinal studies are warranted to confirm these findings.

Fusion expression and anti-Aspergillus flavus activity of a novel inhibitory protein DN-AflR.

The regulatory gene (aflR) encodes AflR, a positive regulator of transcriptional pathway that activates aflatoxin biosynthesis. It has been demonstrated in our laboratory that L-Asp-L-Asn (DN) extracted from Bacillus megaterium inhibited the growth of Aspergillus flavus. We fused gene encoding DN with the gene encoding specific dinuclear zinc finger cluster protein of AflR, then fusion protein competed with the AflS-AflR complex for the AflR binding site and significantly improved anti-A. flavus activity (growth of A. flavus and biosynthesis of aflatoxin B1) of DN. The fusion gene dn-aflR was cloned into pET32a and recombinant plasmid was introduced into Escherichia coli BL21. The highest expression was observed after 10 h induction and fusion protein was purified by affinity chromatography column. Compared with DN, the novel fusion protein DN-AflR significantly inhibited the growth of A. flavus and biosynthesis of aflatoxin B1 (P < 0.05). This study promoted the use of competitive inhibition of fusion proteins to reduce the expression of regulatory genes in the biosynthetic pathway of aflatoxin. Moreover, it provided more supports for deep research and industrialization of such novel anti-A. flavus bio-inhibitors and biological control of microbial contamination.