Circulating trimethylamine N‐oxide and the risk of cardiovascular diseases: a systematic review and meta‐analysis of 11 prospective cohort studies

Circulating trimethylamine N‐oxide (TMAO), a canonical metabolite from gut flora, has been related to the risk of cardiovascular disorders. However, the association between circulating TMAO and the risk of cardiovascular events has not been quantitatively evaluated. We performed a systematic review and meta‐analysis of all available cohort studies regarding the association between baseline circulating TMAO and subsequent cardiovascular events. Embase and PubMed databases were searched for relevant cohort studies. The overall hazard ratios for the developing of cardiovascular events (CVEs) and mortality were extracted. Heterogeneity among the included studies was evaluated with Cochran's Q Test and I² statistics. A random‐effect model or a fixed‐effect model was applied depending on the heterogeneity. Subgroup analysis and meta‐regression were used to evaluate the source of heterogeneity. Among the 11 eligible studies, three reported both CVE and mortality outcome, one reported only CVEs and the other seven provided mortality data only. Higher circulating TMAO was associated with a 23% higher risk of CVEs (HR = 1.23, 95% CI: 1.07–1.42, I² = 31.4%) and a 55% higher risk of all‐cause mortality (HR = 1.55, 95% CI: 1.19–2.02, I² = 80.8%). Notably, the latter association may be blunted by potential publication bias, although sensitivity analysis by omitting one study at a time did not significantly change the results. Further subgroup analysis and meta‐regression did not support that the location of the study, follow‐up duration, publication year, population characteristics or the samples of TMAO affect the results significantly. Higher circulating TMAO may independently predict the risk of subsequent cardiovascular events and mortality.     

Trimethylamine‐N‐oxide promotes brain aging and cognitive impairment in mice

Gut microbiota can influence the aging process and may modulate aging‐related changes in cognitive function. Trimethylamine‐N‐oxide (TMAO), a metabolite of intestinal flora, has been shown to be closely associated with cardiovascular disease and other diseases. However, the relationship between TMAO and aging, especially brain aging, has not been fully elucidated. To explore the relationship between TMAO and brain aging, we analysed the plasma levels of TMAO in both humans and mice and administered exogenous TMAO to 24‐week‐old senescence‐accelerated prone mouse strain 8 (SAMP8) and age‐matched senescence‐accelerated mouse resistant 1 (SAMR1) mice for 16 weeks. We found that the plasma levels of TMAO increased in both the elderly and the aged mice. Compared with SAMR1‐control mice, SAMP8‐control mice exhibited a brain aging phenotype characterized by more senescent cells in the hippocampal CA3 region and cognitive dysfunction. Surprisingly, TMAO treatment increased the number of senescent cells, which were primarily neurons, and enhanced the mitochondrial impairments and superoxide production. Moreover, we observed that TMAO treatment increased synaptic damage and reduced the expression levels of synaptic plasticity‐related proteins by inhibiting the mTOR signalling pathway, which induces and aggravates aging‐related cognitive dysfunction in SAMR1 and SAMP8 mice, respectively. Our findings suggested that TMAO could induce brain aging and age‐related cognitive dysfunction in SAMR1 mice and aggravate the cerebral aging process of SAMP8 mice, which might provide new insight into the effects of intestinal microbiota on the brain aging process and help to delay senescence by regulating intestinal flora metabolites.     

Circulating miR‐145 is associated with plasma high‐sensitivity C‐reactive protein in acute ischemic stroke patients

Stroke is a major cerebrovascular disease threatening human health and life with high morbidity, disability and mortality. We aimed to find effective biomarkers for the early diagnosis on stroke. Nine previously reported stroke‐associated miRNAs (miR‐21, miR‐23a, miR‐29b, miR‐124, miR‐145, miR‐210, miR‐221, miR‐223 and miR‐483‐5p) were measured by quantitative real time‐PCR, and plasma high‐sensitivity C‐reactive protein (hs‐CRP) and serum interleukin 6 (IL‐6), the pro‐inflammation markers in brain injury, were examined by enzyme‐linked immunosorbent assay in 146 acute ischemic stroke patients and 96 healthy blood donors. We found that serum miR‐145 was significantly increased within 24 h after stroke onset and serum miR‐23a and miR‐221 were decreased in patients. Moreover, serum miR‐145 was strong positively correlated with plasma hs‐CRP and moderate positively correlated with serum IL‐6. Meanwhile, serum miR‐23a and miR‐221 were moderate negatively correlated with plasma hs‐CRP but not serum IL‐6. Importantly, the combination of hs‐CRP and serum miR‐145 gained a better sensitivity/spectivity for prediction of acute ischemia stroke (area under receiver operating characteristic curve from 0.794 to 0.896). Conclusively, our preliminary findings indicate that serum miR‐145 upregulated in acute ischemic stroke might be a new biomarker for acute ischemia stroke evaluation. Copyright © 2015 John Wiley & Sons, Ltd.     

Association of Lp‐PLA2 G994T gene polymorphism with risk of ischemic stroke in Chinese population

The association between lipoprotein‐associated phospholipase A2 (Lp‐PLA2) G994T gene polymorphism and the risk of ischemic stroke is unclear. The aim of this study is to investigate the influence of Lp‐PLA2 G994T genetic variant on the pathogenesis of ischemic stroke in Chinese population. A total of 348 patients with a clinical diagnosis of ischemic stroke and 260 gender‐matched control subjects under physical examination were recruited from hospitals and genotyped for G994T gene polymorphism. The results showed that there was a significant difference in the genotype distribution between the two groups and people with GT or TT genotype were associated with the higher risk of ischemic stroke even after adjusting the effects of potential confounding factors. In addition, both ischemic stroke patients and control subjects carrying T allele showed relatively lower Lp‐PLA2 activity and higher oxLDL level. Therefore, Lp‐PLA2 G994T gene polymorphism may be an independent risk factor of ischemic stroke in Chinese population.     

The microvesicle/CD36 complex triggers a prothrombotic phenotype in patients with non‐valvular atrial fibrillation

The mechanisms responsible for platelet activation, the prothrombotic state, in non‐valvular atrial fibrillation (NVAF) are still obscure. Microvesicles (MVs) can transfer various messages to target cells and may be helpful for exploring the detailed mechanisms. We aimed to investigate the possible mechanisms by which proatherogenic factors of NVAF contribute to platelet activation. Two hundred and ten patients with NVAF were stratified as being at ‘low to moderate risk’ or ‘high risk’ for stroke according to the CHADS2 score. Levels of platelet‐derived MVs (PMVs) and platelet activation were examined. CD36‐positive or CD36‐deficient human platelets were stimulated by MVs isolated from NVAF patients with or without various inhibitors in vitro. Levels of PMVs and platelet activation markers enhanced significantly in high‐risk patients. The MVs isolated from plasma of NVAF patients bound to platelet CD36 and activated platelets by phosphorylating the mitogen‐activated protein kinase 4/Jun N‐terminal kinase 2 (MKK4/JNK2) pathways. However, CD36 deficiency protected against MV‐induced activation of platelets. We reveal a possible mechanism of platelet activation in NVAF and suggest that the platelet CD36 might be an effective target in preventing the prothrombotic state in NVAF.     

N‐butylamine functionalized graphene oxide for detection of iron(III) by photoluminescence quenching

An N‐butylamine functionalized graphene oxide nanolayer was synthesized and characterized by ultraviolet (UV)–visible spectrometry, Fourier transform infrared spectroscopy, X‐ray photoelectron spectroscopy, and transmission electron microscopy. Detection of iron(III) based on photoluminescence spectroscopy was investigated. The N‐butylamine functionalized graphene oxide was shown to specifically interact with iron (III), compared with other cationic trace elements including potassium (I), sodium (I), calcium (II), chromium (III), zinc (II), cobalt (II), copper (II), magnesium (II), manganese (II), and molybdenum (VI). The quenching effect of iron (III) on the luminescence emission of N‐butylamine functionalized graphene oxide layer was used to detect iron (III). The limit of detection (2.8 × 10^?6 M) and limit of quantitation (2.9 × 10^?5 M) were obtained under optimal conditions. Copyright © 2015 John Wiley & Sons, Ltd.     

A new quaternary photoluminescence enhancement system of Eu−N‐(o‐vanillin)‐1,8‐diaminonaphthalene−1,10‐phenanthroline−Zn and its application in determining trace amounts of europium and zinc

A new sensitive quaternary photoluminescence enhancement system has been successfully developed to determine trace amounts of Eu³⁺ and Zn²⁺. The photoluminescence intensity of Eu ? N‐(o‐vanilin)‐1,8‐diaminonaphthalene systems was greatly increased by the addition of specific concentrations of 1, 10‐phenanthroline and Zn²⁺. The excitation and emission wavelengths were 274 and 617 nm, respectively. Under optimal system conditions, the photoluminescence intensity showed a linear response toward Eu³⁺ in the range of 5.0 × 10^–6 ~ 2.0 × 10^–5 M with a limit of detection (= 2.2 × 10^–9 M) and the photoluminescence intensity of the system decreased linearly by increasing the Zn²⁺ concentration in the range of 5.0 × 10^–8 ~ 1.0 × 10^–6 M with a limit of detection (= 8.8 × 10^–11 M). This system was successfully applied for the determination of trace amounts of Eu³⁺ in a high purity La₂O₃ matrix and in the synthetic rare earth oxide mixture, and of Zn²⁺ in a high purity Mg(NO₃)₂ · 6H₂O matrix and in synthetic coexisting ionic matrixes. The energy transfer mechanism, photoluminescence enhancement of the system and interference of other lanthanide ions and common coexisting ions were also studied in detail. Copyright © 2013 John Wiley & Sons, Ltd.     

The usefulness of ventricular pacing during atrial fibrillation ablation in a persistent left superior vena cava: A case report

A 69-year-old woman with palpitations was referred to our hospital for a second session of atrial fibrillation (AF) catheter ablation. She had a history of AF ablation including pulmonary vein (PV) isolation and persistent left superior vena cava (PLSVC) isolation. Electrophysiologic studies showed the veno-atrial connections that had recovered. After PV isolation was performed, AF was induced by atrial premature contraction (APC) from the PLSVC, and AF storm occurred. During PLSVC isolation, AF was not induced by APC from the PLSVC. PLSVC isolation continued during sinus rhythm. The elimination of the PLSVC potential was difficult to confirm because of the far-field potential of the left ventricle. Then, we performed right ventricular pacing. The remaining PLSVC potential was identified. After that, the PLSVC isolation was successful during right ventricular pacing. Complications were not observed. The patient had no recurrence of AF thereafter.     

miR‐129‐2‐3p directly targets SYK gene and associates with the risk of ischaemic stroke in a Chinese population

Spleen tyrosine kinase (SYK) gene has been identified as novel susceptibility locus for ischaemic stroke (IS) previously. However, regulation of SYK gene remains unknown in IS. In this study, we aimed to identify miRNAs that might be involved in the development of IS by targeting SYK gene. miRNAs were firstly screened by bioinformatics predicting tool. The expression levels of SYK gene were detected by qRT‐PCR and western blotting, respectively, after miRNA transfection. Luciferase reporter assay was applied to investigate the direct binding between miRNAs and target gene. miRNA levels were detected by miRNA TaqMan assays in the blood cells of 270 IS patients and 270 control volunteers. Results suggest that SYK gene might be a direct target of miR‐129‐2‐3p. The blood level of miR‐129‐2‐3p was significantly lower in IS patients (P < 0.05), and negatively associated with the risk of IS (adjusted OR: 0.88; 95% CI: 0.80‐0.98; P = 0.021) by multivariable logistic regression analysis. The blood levels of SYK gene were significantly higher in IS patients, and miR‐129‐2‐3p expression was negatively correlated with mean platelet volume. In summary, our study suggests that miR‐129‐2‐3p might be involved in the pathogenesis of IS through interrupting SYK expression and the platelet function, and further investigation is needed to explore the underlying mechanism.     

Binding Pockets and Pathways for Dioxygen through the KijD3 N‐Oxygenase in Complex with Flavin Mononucleotide Cofactor and a 3‐Aminoglucose Substrate: Predictions from Molecular Dynamics Simulations

In this work, two protein systems, Kij3D? FMN? AKM? O₂ and Kij3D? FMN? O₂, made of KijD3 N‐oxygenase, flavin mononucleotide (FMN) cofactor, dTDP‐3‐amino‐2,3,6‐trideoxy‐4‐keto‐3‐methyl‐D ‐glucose (AKM) substrate, and dioxygen (O₂), have been assembled by adding a molecule of O₂, and removing (or not) AKM, to crystal data for the Kij3D? FMN? AKM complex. Egress of AKM and O₂ from these systems was then investigated by applying a tiny external random force, in turn, to their center of mass in the course of molecular dynamics in explicit H₂O. It turned out that the wide AKM channel, even when emptied, does not constitute the main route for O₂ egress. Other routes appear to be also viable, while various binding pockets (BPs) outside the active center are prone to trap O₂. By reversing the reasoning, these can also be considered as routes for uptake of O₂ by the protein, before or after AKM uptake, while BPs may serve as reservoirs of O₂. This shows that the small molecule O₂ is capable of permeating the protein by exploiting all nearby interstices that are created on thermal fluctuations of the protein, rather than having necessarily to look for farther, permanent channels.     

Modular pathway engineering of key carbon‐precursor supply‐pathways for improved N‐acetylneuraminic acid production in Bacillus subtilis

N‐acetylneuraminic acid (NeuAc) is widely used as a nutraceutical for facilitating infant brain development, maintaining brain health, and enhancing immunity. Currently, NeuAc is mainly produced by extraction from egg yolk and milk, or via chemical synthesis. However, its low concentration in natural resources and its non‐ecofriendly chemical synthesis result in insufficient NeuAc production and environmental pollution, respectively. In this study, improved NeuAc production was attained via modular pathway engineering of the supply pathways of two key precursors—N‐acetylglucosamine (GlcNAc) and phosphoenolpyruvate (PEP)—and by balancing NeuAc biosynthesis and cell growth in engineered Bacillus subtilis. Specifically, we used a previously constructed GlcNAc‐producing B. subtilis as the initial host for NeuAc biosynthesis. First, we constructed a de novo NeuAc biosynthetic pathway utilizing glucose by coexpressing glucosamine‐6‐phosphate acetyl‐transferase (GNA1), N‐acetylglucosamine 2‐epimerase (AGE), and N‐acetylneuraminic acid synthase (NeuB), resulting in 0.33 g/l NeuAc production. Next, to balance the supply of the two key precursors for NeuAc biosynthesis, modular pathway engineering was performed. The optimal strategy for balancing the GlcNAc module and PEP supply module involved the use of an engineered, unique glucose and malate coutilization pathway in B. subtilis, supplied with both glucose (for the GlcNAc moiety) and malate (for the PEP moiety) at high strength. This led to 1.65 g/L NeuAc production, representing a 5.0‐fold improvement over the existing methods. Furthermore, to enhance the NeuAc yield on cell, glucose and malate coutilization pathways were engineered to balance NeuAc biosynthesis and cell growth via the blocking of glycolysis, the introduction of the Entner–Doudoroff pathway, and the overexpression of the malic enzyme YtsJ. NeuAc titer reached 2.18 g/L, with 0.38 g/g dry cell weight NeuAc yield on cell, which represented a 1.32‐fold and 2.64‐fold improvement over the existing methods, respectively. The strategy of modular pathway engineering of key carbon precursor supply pathways via engineering of the unique glucose‐malate coutilization pathway in B. subtilis should be generically applicable for engineering of B. subtilis for the production of other important biomolecules. Our study also provides a good starting point for further metabolic engineering to achieve industrial production of NeuAc by a Generally Regarded As Safe bacterial strain.     

Protective effect of N‐acetylcysteine against ischemia/reperfusion injury in rat urinary bladders

Ischemia/reperfusion (I/R) injury represents an important cause of bladder contractile dysfunction. One of the major causes leading to this dysfunction is thought to be reactive oxygen species formation. In this study, we investigated the potential benefit of N‐acetylcysteine (NAC), a potent antioxidant that neutralizes free radicals, in a rat model of urinary bladder injury. NAC treatment rescues the reduction of contractile response to I/R injury in a dose‐dependent manner. In addition, all levels of reactive oxygen species, lipid peroxidation, and NADPH‐stimulated superoxide production in the I/R operation + NAC (I/R + NAC) group also decreased compared with a marked increase in the I/R operation + saline (I/R + S) group. Moreover, an in situ fluorohistological approach also showed that NAC reduces the generation of intracellular superoxides enlarged by I/R injury. Together, our findings suggest that NAC has a protective effect against the I/R‐induced bladder contractile dysfunction via radical scavenging property. Copyright © 2013 John Wiley & Sons, Ltd.     

Broad‐substrate screen as a tool to identify substrates for bacterial Gcn5‐related N‐acetyltransferases with unknown substrate specificity

Due to a combination of efforts from individual laboratories and structural genomics centers, there has been a surge in the number of members of the Gcn5‐related acetyltransferasesuperfamily that have been structurally determined within the past decade. Although the number of three‐dimensional structures is increasing steadily, we know little about the individual functions of these enzymes. Part of the difficulty in assigning functions for members of this superfamily is the lack of information regarding how substrates bind to the active site of the protein. The majority of the structures do not show ligand bound in the active site, and since the substrate‐binding domain is not strictly conserved, it is difficult to predict the function based on structure alone. Additionally, the enzymes are capable of acetylating a wide variety of metabolites and many may exhibit promiscuity regarding their ability to acetylate multiple classes of substrates, possibly having multiple functions for the same enzyme. Herein, we present an approach to identify potential substrates for previously uncharacterized members of the Gcn5‐related acetyltransferase superfamily using a variety of metabolites including polyamines, amino acids, antibiotics, peptides, vitamins, catecholamines, and other metabolites. We have identified potential substrates for eight bacterial enzymes of this superfamily. This information will be used to further structurally and functionally characterize them.     

Nuclear internal transcribed spacer‐1 as a sensitive genetic marker for environmental DNA studies in common carp Cyprinus carpio

The recently developed environmental DNA (eDNA) analysis has been used to estimate the distribution of aquatic vertebrates by using mitochondrial DNA (mtDNA) as a genetic marker. However, mtDNA markers have certain drawbacks such as variable copy number and maternal inheritance. In this study, we investigated the potential of using nuclear DNA (ncDNA) as a more reliable genetic marker for eDNA analysis by using common carp (Cyprinus carpio). We measured the copy numbers of cytochrome b (CytB) gene region of mtDNA and internal transcribed spacer 1 (ITS1) region of ribosomal DNA of ncDNA in various carp tissues and then compared the detectability of these markers in eDNA samples. In the DNA extracted from the brain and gill tissues and intestinal contents, CytB was detected at 95.1 ± 10.7 (mean ± 1 standard error), 29.7 ± 1.59 and 24.0 ± 4.33 copies per cell, respectively, and ITS1 was detected at 1760 ± 343, 2880 ± 503 and 1910 ± 352 copies per cell, respectively. In the eDNA samples from mesocosm, pond and lake water, the copy numbers of ITS1 were about 160, 300 and 150 times higher than those of CytB, respectively. The minimum volume of pond water required for quantification was 33 and 100 mL for ITS1 and CytB, respectively. These results suggested that ITS1 is a more sensitive genetic marker for eDNA studies of C. carpio.     

N‐terminal diproline and charge group effects on the stabilization of helical conformation in alanine‐based short peptides: CD studies with water and methanol as solvent

Protein folding problem remains a formidable challenge as main chain, side chain and solvent interactions remain entangled and have been difficult to resolve. Alanine‐based short peptides are promising models to dissect protein folding initiation and propagation structurally as well as energetically. The effect of N‐terminal diproline and charged side chains is assessed on the stabilization of helical conformation in alanine‐based short peptides using circular dichroism (CD) with water and methanol as solvent. A1 (Ac–Pro–Pro–Ala–Lys–Ala–Lys–Ala–Lys–Ala–NH₂) is designed to assess the effect of N‐terminal homochiral diproline and lysine side chains to induce helical conformation. A2 (Ac–Pro–Pro–Glu–Glu–Ala–Ala–Lys–Lys–Ala–NH₂) and A3 (Ac–d Pro–Pro–Glu–Glu–Ala–Ala–Lys–Lys–Ala–NH₂) with N‐terminal homochiral and heterochiral diproline, respectively, are designed to assess the effect of Glu...Lys (i , i  + 4) salt bridge interactions on the stabilization of helical conformation. The CD spectra of A1 , A2 and A3 in water manifest different amplitudes of the observed polyproline II (PPII) signals, which indicate different conformational distributions of the polypeptide structure. The strong effect of solvent substitution from water to methanol is observed for the peptides, and CD spectra in methanol evidence A2 and A3 as helical folds. Temperature‐dependent CD spectra of A1 and A2 in water depict an isodichroic point reflecting coexistence of two conformations, PPII and β‐strand conformation, which is consistent with the previous studies. The results illuminate the effect of N‐terminal diproline and charged side chains in dictating the preferences for extended‐β, semi‐extended PPII and helical conformation in alanine‐based short peptides. The results of the present study will enhance our understanding on stabilization of helical conformation in short peptides and hence aid in the design of novel peptides with helical structures. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Impact of cultivation conditions on N‐glycosylation of influenza virus a hemagglutinin produced in MDCK cell culture

Manufacturers worldwide produce influenza vaccines in different host systems. So far, either fertilized chicken eggs or mammalian cell lines are used. In all these vaccines, hemagglutinin (HA) and neuraminidase are the major components. Both are highly abundant glycoproteins in the viral envelope, and particularly HA is able to induce a strong and protective immune response. The quality characteristics of glycoproteins, such as specific activity, antigenicity, immunogenicity, binding avidity, and receptor‐binding specificity can strongly depend on changes or differences in their glycosylation pattern (potential N‐glycosylation occupancy as well as glycan composition). In this study, capillary gel electrophoresis with laser‐induced fluorescence detection (CGE‐LIF) based glycoanalysis (N‐glycan fingerprinting) was used to determine the impact of cultivation conditions on the HA N‐glycosylation pattern of Madin–Darby canine kidney (MDCK) cell‐derived influenza virus A PR/8/34 (H1N1). We found that adaptation of adherent cells to serum‐free growth has only a minor impact on the HA N‐glycosylation pattern. Only relative abundances of N‐glycan structures are affected. In contrast, host cell adaptation to serum‐free suspension growth resulted in significant changes in the HA N‐glycosylation pattern regarding the presence of specific N‐glycans as well as their abundance. Further controls such as different suppliers for influenza virus A PR/8/34 (H1N1) seed strains, different cultivation scales and vessels in standard or high cell density mode, different virus production media varying in either composition or trypsin activity, different temperatures during virus replication and finally, the impact of β‐propiolactone inactivation resulted—at best—only in minor changes in the relative N‐glycan structure abundances of the HA N‐glycosylation pattern. Surprisingly, these results demonstrate a rather stable HA N‐glycosylation pattern despite various (significant) changes in upstream processing. Only the adaptation of the production host cell line to serum‐free suspension growth significantly influenced HA N‐glycosylation regarding both, the type of attached glycan structures as well as their abundances. Biotechnol. Bioeng. 2013; 110: 1691–1703. © 2013 Wiley Periodicals, Inc.