Author Correction: Bile acid metabolites control TH17 and Treg cell differentiation.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Bile acid metabolites control TH17 and Treg cell differentiation.

Bile acids are abundant in the mammalian gut, where they undergo bacteria-mediated transformation to generate a large pool of bioactive molecules. Although bile acids are known to affect host metabolism, cancer progression and innate immunity, it is unknown whether they affect adaptive immune cells such as T helper cells that express IL-17a (TH17 cells) or regulatory T cells (Treg cells). Here we screen a library of bile acid metabolites and identify two distinct derivatives of lithocholic acid (LCA), 3-oxoLCA and isoalloLCA, as T cell regulators in mice. 3-OxoLCA inhibited the differentiation of TH17 cells by directly binding to the key transcription factor retinoid-related orphan receptor-γt (RORγt) and isoalloLCA increased the differentiation of Treg cells through the production of mitochondrial reactive oxygen species (mitoROS), which led to increased expression of FOXP3. The isoalloLCA-mediated enhancement of Treg cell differentiation required an intronic Foxp3 enhancer, the conserved noncoding sequence (CNS) 3; this represents a mode of action distinct from that of previously identified metabolites that increase Treg cell differentiation, which require CNS1. The administration of 3-oxoLCA and isoalloLCA to mice reduced TH17 cell differentiation and increased Treg cell differentiation, respectively, in the intestinal lamina propria. Our data suggest mechanisms through which bile acid metabolites control host immune responses, by directly modulating the balance of TH17 and Treg cells.

Bidirectional modulation of HIF-2 activity through chemical ligands.

Hypoxia-inducible factor-2 (HIF-2) is a heterodimeric transcription factor formed through dimerization between an oxygen-sensitive HIF-2α subunit and its obligate partner subunit ARNT. Enhanced HIF-2 activity drives some cancers, whereas reduced activity causes anemia in chronic kidney disease. Therefore, modulation of HIF-2 activity via direct-binding ligands could provide many new therapeutic benefits. Here, we explored HIF-2α chemical ligands using combined crystallographic, biophysical, and cell-based functional studies. We found chemically unrelated antagonists to employ the same mechanism of action. Their binding displaced residue M252 from inside the HIF-2α PAS-B pocket toward the ARNT subunit to weaken heterodimerization. We also identified first-in-class HIF-2α agonists and found that they significantly displaced pocket residue Y281. Its dramatic side chain movement increases heterodimerization stability and transcriptional activity. Our findings show that despite binding to the same HIF-2α PAS-B pocket, ligands can manifest as inhibitors versus activators by mobilizing different pocket residues to allosterically alter HIF-2α-ARNT heterodimerization.

Structural integration in hypoxia-inducible factors.

The hypoxia-inducible factors (HIFs) coordinate cellular adaptations to low oxygen stress by regulating transcriptional programs in erythropoiesis, angiogenesis and metabolism. These programs promote the growth and progression of many tumours, making HIFs attractive anticancer targets. Transcriptionally active HIFs consist of HIF-α and ARNT (also called HIF-1ß) subunits. Here we describe crystal structures for each of mouse HIF-2α-ARNT and HIF-1α-ARNT heterodimers in states that include bound small molecules and their hypoxia response element. A highly integrated quaternary architecture is shared by HIF-2α-ARNT and HIF-1α-ARNT, wherein ARNT spirals around the outside of each HIF-α subunit. Five distinct pockets are observed that permit small-molecule binding, including PAS domain encapsulated sites and an interfacial cavity formed through subunit heterodimerization. The DNA-reading head rotates, extends and cooperates with a distal PAS domain to bind hypoxia response elements. HIF-α mutations linked to human cancers map to sensitive sites that establish DNA binding and the stability of PAS domains and pockets.

Tanshinone IIA Can Inhibit Angiotensin II-Induced Proliferation and Autophagy of Vascular Smooth Muscle Cells via Regulating the MAPK Signaling Pathway.

To examine the effect of tanshinone IIA on Angiotensin II (Ang II)-induced proliferation and autophagy in vascular smooth muscle cells (VSMCs) and the related mechanism. VSMCs were treated with Ang II with or without tanshinone IIA (1, 5 and 10 µg/mL), and the proliferation, apoptosis in cells with different treatment were examined by methylthiazolyl tetrazolium (MTT) and flow cytometry methods. Moreover, the expression of autophagy related proteins and mitogen-activated protein kinase (MAPK) signaling molecules were examined by RT-quantitative (q)PCR and Western blot methods. Ang II induced significantly increase in the proliferation and autophagy of VSMCs, and the MAPK signaling was activated. Tanshinone IIA can attenuate Ang II-induced effects via down-regulating the MAPK signaling pathway. Tanshinone IIA can inhibit Ang II-induced proliferation and autophagy of VSMCs via regulating the MAPK signaling pathway.

Doc2b serves as a scaffolding platform for concurrent binding of multiple Munc18 isoforms in pancreatic islet ß-cells.

Biphasic glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells involves soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor (SNARE) protein-regulated exocytosis. SNARE complex assembly further requires the regulatory proteins Munc18c, Munc18-1 and Doc2b. Munc18-1 and Munc18c are required for first- and second-phase GSIS respectively. These distinct Munc18-1 and Munc18c roles are related to their transient high-affinity binding with their cognate target (t-)SNAREs, Syntaxin 1A and Syntaxin 4 respectively. Doc2b is essential for both phases of GSIS, yet the molecular basis for this remains unresolved. Because Doc2b binds to Munc18-1 and Munc18c via its distinct C2A and C2B domains respectively, we hypothesized that Doc2b may provide a plasma membrane-localized scaffold/platform for transient docking of these Munc18 isoforms during GSIS. Towards this, macromolecular complexes composed of Munc18c, Doc2b and Munc18-1 were detected in ß-cells. In vitro interaction assays indicated that Doc2b is required to bridge the interaction between Munc18c and Munc18-1 in the macromolecular complex; Munc18c and Munc18-1 failed to associate in the absence of Doc2b. Competition-based GST-Doc2b interaction assays revealed that Doc2b could simultaneously bind both Munc18-1 and Munc18c. Hence these data support a working model wherein Doc2b functions as a docking platform/scaffold for transient interactions with the multiple Munc18 isoforms operative in insulin release, promoting SNARE assembly.

[Long-term outcomes and factors for predicting ventricular arrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia].

OBJECTIVE: To evaluate factors for predicting ventricular arrhythmia, the clinical effect of drugs on patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), and their long-term outcomes. METHODS: Six patients diagnosed with CPVT underwent a series of electrocardiograms and 24-hour Holter monitoring. ß-blockers were recommended for all patients, while some patients were also prescribed propafenone and 1 patient underwent catheter-based renal sympathetic denervation (RDN). The characteristics of electrocardiogram, arrhythmia and long-term outcomes were monitored. RESULTS: Syncope episodes did not occur any longer in 1 patient on ß-blocker, but recurred in 3 other patients and 2 patients died (one due to his cessation of metoprolol for 3 months). Inverted and/or bifid T waves and abnormal U wave were observed in the precordial leads. T wave alternans was observed in 4 patients in the precordial leads. These abnormal electrocardiogram features disappeared or diminished with ß-blocker treatment. All spontaneous episodes of ventricular tachycardia occurred prior to sinus tachycardia and frequent polymorphic premature ventricular contractions. CONCLUSIONS: Bifid and/or inverted T waves, T wave alternans and abnormal U waves together with sinus tachycardia and frequent premature ventricular contractions are indicator for predicting ventricular arrhythmia and assessing the effect of ß-blockers. Compliance with ß-blocker treatment is a strong indicator of outcome.

Zinc-doped and biochar support strategies to enhance the catalytic activity of CuFe2O4 to persulfate for crystal violet degradation.

Sulfate radicals-based Fenton-like technology has placed more emphasis on effectively dealing with the threat of dye wastewater. In this work, the Zn-doped CuFe2O4@biochar composite (Cu0.9Zn0.1Fe2O4@BC) was prepared through the convenient sol-gel pyrolysis process and applied as heterogeneous persulfate (PS) activator for crystal violet (CV) degradation. The crystal morphology and physicochemical properties of Cu0.9Zn0.1Fe2O4@BC were investigated by scanning electron microscope (SEM), X-ray diffractometer (XRD), vibrating sample magnetometer (VSM), Brunauer-Emmett-Teller method (BET), and X-ray photoelectron spectroscopy (XPS). The morphology of the catalyst changed before and after Zn doping. The crystallite size, lattice constant, saturation magnetization, and oxygen vacancy content increased after doping Zn. Compared with CuFe2O4@BC, the CV degradation efficiency of Cu0.9Zn0.1Fe2O4@BC activating PS increased from 87.7 to 96.9%, and the corresponding reaction rate constant increased by about 3.69 times. The effect of experimental conditions was systematically studied on the degradation progress. The degradation efficiency of CV was 91% after five times cycle experiments. Multiple experiments indicated that SO4•-, •OH and O2•- predominated for CV degradation. The degradation mechanism of CV in the Cu0.9Zn0.1Fe2O4@BC/PS system involved both free radical (SO4•-, •OH and O2•-) and non-free radical pathways (electron transfer). The possible degradation pathways were investigated according to the ultra-performance liquid chromatography mass spectrometry (UPLC-MS) analysis of degradation intermediates. The result showed that Cu0.9Zn0.1Fe2O4@BC have an excellent catalyst performance, which provides a new strategy for improving catalytic activity.

[Clinical features of six patients with catecholaminergic polymorphic ventricular tachycardia].

OBJECTIVE: To describe the clinical features of 6 patients with catecholaminergic polymorphic ventricular tachycardia. METHODS: Clinical data including signs and symptoms, electrocardiograms, Holter monitoring electrocardiograms and echocardiography was analyzed. Definite diagnosis was made based on the mutations of RYR2 and CASQ2. RESULTS: From July 2002 to March 2010, 6 consecutive patients referred to our center because of syncope [4 males, mean age (13.0 ± 4.2) years] were diagnosed with CPVT by clinical evaluation and genetic testing. Their electrocardiograms showed T waves with notch or bimodal and tall U waves in right chest leads. There was no J wave, no ST-segment deviation, no prolongation or shortening of QT interval. We captured the so-called "bidirectional and(or) polymorphic ventricular tachycardia (bVT and pVT)" in 2 out of 6 patients by ECG, in 5 out of 6 patients by 24-hours Holter monitor, in 3 out of 6 patients by exercise test. All patients received ß blockers and no syncope occurred during the 3 months follow-up after discharge from hospital. CONCLUSIONS: CPVT is an inherited cardiac channelopathy characterized by syncope and(or) sudden death relatived to motion. The ECG shows T wave alteration and tall U wave in right chest leads. The mode of its onset is bVT and(or) pVT, and can be captured by Holter easily. ß blocker is a safe and effective remedy for suppressing its attack.

Bathroom modifications among community-dwelling older adults who experience falls in the United States: A cross-sectional study.

Falls impose substantial health and economic burdens on older adults. Over half of falls in older adults occur at home, with many involving bathroom areas. Limited information is available on the presence of bathroom modifications for those who experience them. Therefore, we examined factors associated with bathroom modifications among older adults with at least one fall in the United States. We analysed the nationally representative 2016 Medicare Current Beneficiary Survey Public Use File of Medicare beneficiaries aged ≥65 years with ≥1 fall (n = 2,404). A survey-weighted logistic model was used to examine associations between bathroom modifications and factors including socio-demographic characteristics, health-related conditions, and fear of falling. Among Medicare beneficiaries with ≥1 fall, 55.5% had bathroom modifications and 50.1% had repeated falls (≥2 falls). Approximately 40.2% of those with repeated falls had no bathroom modifications. In the adjusted model, non-Hispanic Blacks (odds ratio [OR] = 0.38; p < 0.001) and Hispanics (OR = 0.64; p = 0.039) had lower odds of having bathroom modifications than non-Hispanic Whites. Fear of falling and activities of daily living limitations had incremental impacts on having bathroom modifications. This study highlights the need to improve disparities in bathroom modifications for non-Hispanic Black and Hispanic Medicare beneficiaries, including those with repeated falls. With the aging population and growing number of older minorities in the United States, reducing these disparities is vital for fall prevention efforts and aging-in-place.

Identification of oleoylethanolamide as an endogenous ligand for HIF-3α.

Hypoxia-inducible factors (HIFs) are α/ß heterodimeric transcription factors modulating cellular responses to the low oxygen condition. Among three HIF-α isoforms, HIF-3α is the least studied to date. Here we show that oleoylethanolamide (OEA), a physiological lipid known to regulate food intake and metabolism, binds selectively to HIF-3α. Through crystallographic analysis of HIF-3 α/ß heterodimer in both apo and OEA-bound forms, hydrogen-deuterium exchange mass spectrometry (HDX-MS), molecular dynamics (MD) simulations, and biochemical and cell-based assays, we unveil the molecular mechanism of OEA entry and binding to the PAS-B pocket of HIF-3α, and show that it leads to enhanced heterodimer stability and functional modulation of HIF-3. The identification of HIF-3α as a selective lipid sensor is consistent with recent human genetic findings linking HIF-3α with obesity, and demonstrates that endogenous metabolites can directly interact with HIF-α proteins to modulate their activities, potentially as a regulatory mechanism supplementary to the well-known oxygen-dependent HIF-α hydroxylation.

Characterization of Microbial Community in Cold-Chain Hairtail Fish by High-Throughput Sequencing Technology.

ABSTRACT: High-throughput DNA sequencing with the Illumina MiSeq platform was used to analyze the microbial communities in hairtail (Trichiurus haumela) muscle samples to study the diversity and dynamic changes in these communities during cold-chain circulation of these fish. The richness and diversity of the microbial community in hairtail muscle had a transient decline from 0 to 24 h and decreased after the first rise from 24 to 216 h. The diversity and richness of bacteria in cold-chain hairtail reached maximum at 168 h. The Shannon and Simpson diversity indices of the bacteria were 2.96 and 0.16, respectively, and their ACE and Chao1 richness indices were 254.84 and 155.10, respectively. The dominant bacteria belonged to phylum Proteobacteria, class Gammaproteobacteria, order Pseudomonadales, family Pseudomonadaceae, and genus Pseudomonas, and their relative abundances were 80.52, 72.11, 76.68, 23.25, and 53.50%, respectively. These results provide a basis for exploring how to maintain the freshness and predict the shelf life of hairtail.

Simultaneous Detection of Seven Alternaria Toxins in Mixed Fruit Puree by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry Coupled with a Modified QuEChERS.

The presence of Alternaria toxins (ATs) in fruit purees may cause potential harm to the life and health of consumers. As time passes, ATs have become the key detection objects in this kind of food. Based on this, a novel and rapid method was established in this paper for the simultaneous detection of seven ATS (tenuazonic acid, alternariol, alternariol monomethyl ether, altenuene, tentoxin, altenusin, and altertoxin I) in mixed fruit purees using ultra-high performance liquid chromatography-tandem mass spectrometry. The sample was prepared using the modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method to complete the extraction and clean-up steps in one procedure. In this QuEChERS method, sample was extracted with water and acetonitrile (1.5% formic acid), then salted out with NaCl, separated on an ACQUITY UPLC BEH C18 with gradient elution by using acetonitrile and 0.1% formic acid aqueous as eluent, and detected by UPLC-MS/MS under positive (ESI+) and negative (ESI-) electrospray ionization and MRM models. Results showed that the seven ATs exhibited a good linearity in the concentration range of 0.5-200 ng/mL with R2 > 0.9925, and the limits of detection (LODs) of the instrument were in the range of 0.18-0.53 µg/kg. The average recoveries ranged from 79.5% to 106.7%, with the relative standard deviations (RSDs) no more than 9.78% at spiked levels of 5, 10, and 20 µg/kg for seven ATs. The established method was applied to the determination and analysis of the seven ATs in 80 mixed fruit puree samples. The results showed that ATs were detected in 31 of the 80 samples, and the content of ATs ranged from 1.32 µg/kg to 54.89 µg/kg. Moreover, the content of TeA was the highest in the detected samples (23.32-54.89 µg/kg), while the detection rate of Ten (24/31 samples) was higher than the other ATs. Furthermore, the other five ATs had similar and lower levels of contamination. The method established in this paper is accurate, rapid, simple, sensitive, repeatable, and stable, and can be used for the practical determination of seven ATs in fruit puree or other similar samples. Moreover, this method could provide theory foundation for the establishment of limit standard of ATs and provide a reference for the development of similar detection standard methods in the future.

Atorvastatin protects against angiotensin II-induced injury and dysfunction in human umbilical vein endothelial cells through bradykinin 2 receptors.

The morphological and functional integrity of the endothelial cell (EC) is compromised in many cardiovascular diseases such as atherosclerosis, hypertension, and diabetes. Angiotensin II (Ang II) plays important roles in the initiation and progression of these diseases. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) may have cholesterol-independent pleiotropic effects on preventing the EC injury and dysfunction that occurs in these diseases, and the protective effects may relate to bradykinin 2 receptors (B2Rs). Our study was designed to test the hypothesis that atorvastatin, via B2Rs, protects the viability and function of EC exposed to Ang II independent of hemodynamics. The experimental results showed that the cytotoxic effects of Ang II on human umbilical vein endothelial cells were significantly ameliorated by atorvastatin pretreatment (LDH tests, MTT assay, and propdium iodide (PI)/Annexin V-stating analysis), and atorvastatin treatment simultaneously enhanced expression of endothelial nitric oxide synthase and yielded of nitric oxide (NO) and cyclic guanosine monophosphate, but both effects were attenuated by the B2Rs antagonist HOE-140. This study proves the hypothesis and may be pertinent to the complex mechanism of action of statins explaining their long-term beneficial effects in maintaining the morphological and functional integrity of vascular ECs.

Expression and characterization of Mycobacterium tuberculosis methionine aminopeptidase type 1a.

Methionine aminopeptidase (MetAP) carries out the cotranslational N-terminal methionine excision and is essential for bacterial survival. Mycobacterium tuberculosis expresses two MetAPs, MtMetAP1a and MtMetAP1c, at different levels in growing and stationary phases, and both are potential targets to develop novel antitubercular therapeutics. Recombinant MtMetAP1a was purified as an apoenzyme, and metal binding and activation were characterized with an activity assay using a fluorogenic substrate. Ni(II), Co(II) and Fe(II) bound tightly at micromolar concentrations, and Ni(II) was the most efficient activator for the MetAP-catalyzed substrate hydrolysis. Although the characteristics of metal binding and activation are similar to MtMetAP1c we characterized before, MtMetAP1a was significantly more active, and more importantly, a set of inhibitors displayed completely different inhibitory profiles on the two mycobacterial MetAPs in both potency and metalloform selectivity. The differences in catalysis and inhibition predicted the significant differences in active site structure.

Determination of binding affinity of metal cofactor to the active site of methionine aminopeptidase based on quantitation of functional enzyme.

Determination of metal affinity to the active site of metalloenzymes constitutes an integral part in the understanding of enzyme catalysis and regulation. Nonlinear curve fitting of metal titration curves using the multiple independent binding sites (MIBS) model was adapted to determine K(D) values based on functional enzyme concentrations. This approach provides a more accurate evaluation of K(D) compared with existing methods that are based on total protein concentrations. We applied this concept to methionine aminopeptidase from Mycobacterium tuberculosis and showed that it is a monometalated enzyme with a K(D) of 0.13 microM for Co(2+).

Early auditory deprivation alters expression of NMDA receptor subunit NR1 mRNA in the rat auditory cortex.

The expression of NMDA receptor NR1 subunit mRNA was studied in rat auditory cortex (AC) on different postnatal days using digoxigenin-labeled oligonucleotide probes. The results showed that NR1 expression increased from birth to postnatal day 35 (P35) and remained constant until P56. The most significant increases occurred between P7 and P14. Changes in NR1 mRNA expression in rats subjected to monaural hearing deprivation on P7, P21, P35, and P49 were examined on P56. Between P7 and P21, when the rat auditory system was still in a critical period of development, NR1 mRNA expression was lower in the contralateral AC, which received auditory signals from the plugged ear, than in the ipsilateral AC. However, no significant difference was observed between the rats deprived of hearing on P35 and those deprived of hearing on P42, the end of the critical period of auditory development. These results showed that monaural hearing deprivation during early postnatal development was associated with decreased NR1 mRNA expression in the contralateral AC and suggested the involvement of NR1 in auditory function during development. They also indicated that, during postnatal development, environmental factors changed the functional plasticity of neurons in the AC through NR1 receptor expression. Taken together, these findings provide a possible underlying mechanism for the development of postnatal auditory function.

Sick sinus syndrome associated with hypopituitarism: a case report and literature review.

Though an association between autoimmune diseases and sick sinus syndrome has been reported, there has been no report on the association of hypopituitarism and sick sinus syndrome. Herein, we provide the first case report of hypopituitarism accompanying sick sinus syndrome in a 51-year-old woman presented to our hospital with syncope due to cardiac arrest. The patient was successfully managed by pacemaker installation and hormone replacement therapy.

Serum miR-210 and miR-30a expressions tend to revert to fetal levels in Chinese adult patients with chronic heart failure.

BACKGROUND: MicroRNAs (miRNAs) are widely involved in the process of chronic heart failure (HF), which is characterized by reactivation of the fetal gene program. Here, we examined whether the serum expression levels of some HF-related miRNAs in adult HF patients would tend to revert to fetal levels. METHODS AND RESULTS: Serum was obtained from the peripheral venous blood of 22 HF patients, 18 asymptomatic controls, and the umbilical venous blood of 9 fetuses from 9 independent parturitions. Serum pools of the three groups were initially screened against 40 known HF-associated miRNAs via quantitative reverse transcriptase polymerase chain reaction. Twenty-seven miRNAs were stably expressed in the serum pools. Nine miRNAs showed similar expression levels in the HF and fetus groups compared to the controls, two of which (miR-210, miR-30a) were significantly up-regulated in both groups. These miRNAs showed high diagnostic accuracy and correlations with blood N-terminal prohormone of brain natriuretic peptide, identifying them as potential biomarkers for HF. Putative targets of the miRNAs were predicted with online software programs, and the Kyoto Encyclopedia of Genes and Genomes pathway analysis was employed to identify miRNA-regulated functional modules. In particular, miR-210 seemed to be more closely related than miR-30a to the pathological mechanisms of HF, including the calcium signaling, vascular smooth muscle contraction, transforming growth factor-ß signaling, and aldosterone-regulated sodium reabsorption pathways. CONCLUSION: The serum expression levels of some HF-related miRNAs in HF patients tended towards fetal levels. Among them, miR-210 and miR-30a were elevated in the HF and fetus groups.

Evidence of a role of inositol polyphosphate 5-phosphatase INPP5E in cilia formation in zebrafish.

Inositol phosphatases are important regulators of cell signaling and membrane trafficking. Mutations in inositol polyphosphate 5-phosphatase, INPP5E, have been identified in Joubert syndrome, a rare congenital disorder characterized by midbrain malformation, retinitis pigmentosa, renal cysts, and polydactyly. Previous studies have implicated primary cilia abnormalities in Joubert syndrome, yet the role of INPP5E in cilia formation is not well understood. In this study, we examined the function of INPP5E in cilia development in zebrafish. Using specific antisense morpholino oligonucleotides to knockdown Inpp5e expression, we observed phenotypes of microphthalmia, pronephros cysts, pericardial effusion, and left-right body axis asymmetry. The Inpp5e morphant zebrafish exhibited shortened and decreased cilia formation in the Kupffer's vesicle and pronephric ducts as compared to controls. Epinephrine-stimulated melanosome trafficking was delayed in the Inpp5e zebrafish morphants. Expression of human INPP5E expression rescued the phenotypic defects in the Inpp5e morphants. Taken together, we showed that INPP5E is critical for the cilia development in zebrafish.