Ablation of Mto1 in zebrafish exhibited hypertrophic cardiomyopathy manifested by mitochondrion RNA maturation deficiency.

Deficient maturations of mitochondrial transcripts are linked to clinical abnormalities but their pathophysiology remains elusive. Previous investigations showed that pathogenic variants in MTO1 for the biosynthesis of τm5U of tRNAGlu, tRNAGln, tRNALys, tRNATrp and tRNALeu(UUR) were associated with hypertrophic cardiomyopathy (HCM). Using mto1 knock-out(KO) zebrafish generated by CRISPR/Cas9 system, we demonstrated the pleiotropic effects of Mto1 deficiency on mitochondrial RNA maturations. The perturbed structure and stability of tRNAs caused by mto1 deletion were evidenced by conformation changes and sensitivity to S1-mediated digestion of tRNAGln, tRNALys, tRNATrp and tRNALeu(UUR). Notably, mto1KO zebrafish exhibited the global decreases in the aminoacylation of mitochondrial tRNAs with the taurine modification. Strikingly, ablated mto1 mediated the expression of MTPAP and caused the altered polyadenylation of cox1, cox3, and nd1 mRNAs. Immunoprecipitation assay indicated the interaction of MTO1 with MTPAP related to mRNA polyadenylation. These alterations impaired mitochondrial translation and reduced activities of oxidative phosphorylation complexes. These mitochondria dysfunctions caused heart development defects and hypertrophy of cardiomyocytes and myocardial fiber disarray in ventricles. These cardiac defects in the mto1KO zebrafish recapitulated the clinical phenotypes in HCM patients carrying the MTO1 mutation(s). Our findings highlighted the critical role of MTO1 in mitochondrial transcript maturation and their pathological consequences in hypertrophic cardiomyopathy.

Home-produced eggs: An important pathway of methylmercury exposure for residents in mercury mining areas, southwest China.

In light of the documented elevated concentrations of total mercury (Hg) and methylmercury (MeHg) in poultry originating from Hg-contaminated sites, a knowledge gap persists regarding the levels of Hg found in home-produced eggs (HPEs) and the associated dietary exposure risks in regions affected by Hg mining. To address this knowledge gap, a comprehensive investigation was undertaken with the primary objectives of ascertaining the concentrations of THg and MeHg in HPEs and evaluating the potential hazards associated with the consumption of eggs from the Wanshan Hg mining area in Southwest China. The results showed that THg concentrations in HPEs varied within a range of 10.5-809 ng/g (with a geometric mean (GM) of 64.1 ± 2.7 ng/g), whereas MeHg levels spanned from 1.3 to 291 ng/g (GM, 23.1 ± 3.4 ng/g). Remarkably, in half of all eggs, as well as those collected from regions significantly impacted by mining activities, THg concentrations exceeded the permissible maximum allowable value for fresh eggs (50 ng/g). Consumption of these eggs resulted in increased exposure risks associated with THg and MeHg, with GM values ranging from 0.024 to 0.17 µg/kg BW/day and 0.0089-0.066 µg/kg BW/day, respectively. Notably, the most substantial daily dosage was observed among children aged 2-3 years. The study found that consuming HPEs could result in a significant IQ reduction of 34.0 points for the whole mining area in a year. These findings highlight the potential exposure risk, particularly concerning MeHg, stemming from the consumption of local HPEs by residents in mining areas, thereby warranting serious consideration within the framework of Hg exposure risk assessment in mining locales.

Sub-chronic exposure to hexaconazole affects the lipid metabolism of rats through mTOR-PPAR-γ/SREBP1 signaling pathway mediated by oxidative stress.

Hexaconazole (Hex) is a widely used and high frequency detected triazole fungicide in agricultural products and environment which may pose potential toxicity to the nontargeted organisms. Hex had been reported to affect lipid homeostasis while the mechanism was undefined. This study aims to explore the characteristic lipidomic profiles and clarify the underlying signaling pathways of Hex-induced lipid metabolism disorder in rat liver. The results showed that sub-chronic exposure to environmental related concentrations of Hex caused histopathological changes, oxidative stress, fat accumulation, lipid biochemical parameter increase in rats. Moreover, the untargeted lipidomic analysis showed that the levels of TAG, PC, and PE and the pathway of glycerophospholipid metabolism were heavily altered by Hex. We further analyzed the lipid metabolism related genes and proteins which revealed that Hex exposure increased amount of lipogenesis by activating oxidative stress-mediated mTOR-PPAR-γ/SREBP1 signaling pathways. The imbalance of lipid homeostasis induced by Hex exposure might further lead to obesity, cardiovascular diseases (CVDs), and hyperlipidemia. Our results provided systematic and comprehensive evidence for the mechanism of Hex-induced lipid metabolism disorder at environmental concentrations and supplied a certain basis for its health risks assessment.

Chitosan Enhances Low-Dosage Difenoconazole to Efficiently Control Leaf Spot Disease in Pseudostellaria heterophylla (Miq.) Pax.

Pseudostellaria heterophylla (Miq.) Pax is a popular clinical herb and nutritious health food. However, leaf spot disease caused by fungal pathogens frequently occurs and seriously influences the growth, quality, and yield of P. heterophylla. In this work, the field control roles of difenoconazole, chitosan, and their combination in the leaf spot disease in P. heterophylla and their effects on the disease resistance, photosynthetic capacity, medicinal quality, and root yield of P. heterophylla are investigated. The results manifest that 37% difenoconazole water-dispersible granule (WDG) with 5000-time + chitosan 500-time dilution liquid had a superior control capacity on leaf spot disease with the control effects of 91.17%~88.19% at 15~30 days after the last spraying, which significantly (p < 0.05) exceeded that of 37% difenoconazole WDG 3000-time dilution liquid and was significantly (p < 0.01) higher than that of 37% difenoconazole WDG 5000-time dilution liquid, chitosan 500-time dilution liquid, or chitosan 1000-time dilution liquid. Simultaneously, this combination could more effectively enhance the disease resistance, photosynthetic capacity, medicinal quality, and tuberous root yield of P. heterophylla compared to when these elements were applied alone, as well as effectively reduce difenoconazole application. This study emphasizes that chitosan combined with a low dosage of difenoconazole can be proposed as a green, efficient, and alternative formula for controlling leaf spot disease in P. heterophylla and enhancing its resistance, photosynthesis, quality, and yield.

Asymmetrical effects of deafness-associated mitochondrial DNA 7516delA mutation on the processing of RNAs in the H-strand and L-strand polycistronic transcripts.

In this report, we investigated the molecular mechanism underlying a deafness-associated m.7516delA mutation affecting the 5' end processing sites of mitochondrial tRNAAsp and tRNASer(UCN). An in vitro processing experiment demonstrated that m.7516delA mutation caused the aberrant 5' end processing of tRNASer(UCN) and tRNAAsp precursors, catalyzed by RNase P. Using cytoplasmic hybrids (cybrids) derived from one hearing-impaired Chinese family bearing the m.7516delA mutation and control, we demonstrated the asymmetrical effects of m.7516delA mutation on the processing of tRNAs in the heavy (H)-strand and light (L)-strand polycistronic transcripts. Specially, the m.7516delA mutation caused the decreased levels of tRNASer(UCN) and downstream five tRNAs, including tRNATyr from the L-strand transcripts and tRNAAsp from the H-strand transcripts. Strikingly, mutant cybrids exhibited the lower level of COX2 mRNA and accumulation of longer and uncleaved precursors of COX2 from the H-strand transcripts. Aberrant RNA metabolisms yielded variable reductions in the mitochondrial proteins, especially marked reductions in the levels of ND4, ND5, CO1, CO2 and CO3. The impairment of mitochondrial translation caused the proteostasis stress and respiratory deficiency, diminished ATP production and membrane potential, increased production of reactive oxygen species and promoted apoptosis. Our findings provide new insights into the pathophysiology of deafness arising from mitochondrial tRNA processing defects.

Dapagliflozin, sildenafil and their combination in monocrotaline-induced pulmonary arterial hypertension.

BACKGROUND: Dapagliflozin, a selective inhibitor of sodium-glucose cotransporter 2 (SGLT2), can reduce cardiovascular events and mortality in patients with heart failure. A number of mechanisms have been proposed to explain the beneficial effects of SGLT2 inhibitors. The purpose of this study was to determine whether dapagliflozin can improve pulmonary vascular remodelling and the efficacy of dapagliflozin as an add-on therapy to sildenafil in rats with pulmonary arterial hypertension (PAH). METHODS: A monocrotaline (MCT)-induced PAH rat model was used in our study. MCT-injected rats were randomly divided into four groups and treated for 3 weeks with daily per os treatment with vehicle, dapagliflozin (1 mg/kg/day), sildenafil (25 mg/kg/day), or a combination of dapagliflozin (1 mg/kg/day) and sildenafil (25 mg/kg/day). Haemodynamic measurements, histological analysis, enzyme-linked immunosorbent assay and western blotting analysis were employed to detect the changes in PAH rats after treatments. RESULTS: Dapagliflozin significantly attenuated MCT-induced increases in right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH) in PAH rats. Dapagliflozin effectively decreased the thickening of pulmonary artery media and decreased the muscularization of pulmonary arterioles in PAH rats. Moreover, dapagliflozin attenuated nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome activation in lung tissues and the levels of interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) in plasma. However, dapagliflozin as an add-on therapy to sildenafil in rats with PAH did not show a more pronounced beneficial effect on right ventricular systolic pressure and pulmonary vascular remodelling in MCT rats than sildenafil alone. CONCLUSIONS: Dapagliflozin reduces right ventricular systolic pressure and pulmonary vascular remodelling in a rat model of PAH. However, combination therapy with dapagliflozin and sildenafil was not more effective than monotherapy with sildenafil in PAH rats.

Distributions of Heavy Metals in Rice and Corn and Their Health Risk Assessment in Guizhou Province.

Long-term exposure to heavy metals from high geological background area may lead to varieties of diseases. Therefore, risk assessment from agricultural products in these areas was crucial to ensure the health of customers. However, the effects of geological background on distributions of heavy metals and their accumulation in plant remain unclear. This study aimed to determine the distributions of mercury (Hg), cadmium (Cd), arsenic (As), lead (Pb), chromium (Cr) and copper (Cu) in 1036 corn and rice samples collected from 9 locations in Guizhou province and to evaluate their health risks. The concentrations of Hg, Cd, As, Pb, Cr and Cu in these two crops were analyzed by inductively coupled plasma-mass spectrometry (ICP-MS), and their health risk were estimated by the target risk quotient approaches. Results showed that the mean concentrations of Hg, Cd, As, Pb, Cr and Cu in corn and rice were 0.01, 0.07, 0.06, 0.27, 0.56 and 1.86 mg/kg which were lower than their respective maximum allowable concentrations (MAC) of 0.02, 0.20, 0.70, 0.20, 0.50 and 10.00 mg/kg except Pb and Cr. The concentrations of Cr and Cu in corn were higher than in rice while Cd, As and Pb in rice were higher than in corn. Moreover, the distributions of Hg, Cd and Cu in corn and rice samples were mainly observed from QDN located at southeast of Guizhou province while Pb, As and Cr were most detected at ZY, QXN and BJ areas, southwest zone. The hazard indices (HIs) values for corn and rice were 0.20 and 2.61. The high HIs (> 1) in rice indicated that the health risk of heavy metals in rice was relatively high and Pb was the major component that attributed to the risk, followed by Cd. These results could provide a reference for the distributions of heavy metals in agricultural products in Guizhou province under crop cultivation conditions, and to provide scientific basis for health risk assessment and ensure food safety.

The discovery of combined toxicity effects and mechanisms of hexaconazole and arsenic to mice based on untargeted metabolomics.

The high detected frequencies of hexaconazole (Hex) and arsenic (As) increased the probabilities of their co-existence in agricultural products. However, the combined toxicity effect and mechanism of action for these two pollutants were still unclear. In this study, an untargeted metabolomics method with ultra high performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS) was developed to monitor the changes of endogenous metabolites and metabolism pathways in mice liver. Our study revealed that significant differences in metabolomics profiles were observed after Hex, As, and Hex+As exposure for 90 d. Hex exposure altered 54 metabolites and 11 pathways significantly which were mainly lipid-related. For As exposure, 63 metabolites and 9 pathways were affected most of which were amino acid-related. Hex+As induced 93 metabolites changes with 34% was lipids and lipid-like molecules and 22% was organic acids and derivatives. Hex+As exposure shared the pathways that altered by Hex and As indicated that the interaction of Hex and As might be independent action. The results of this study could provide an important insight for understanding the mechanism of combined toxicity for Hex and As and be helpful for evaluating their health risk to human.

Insights into the Genetic Variations of Human Cytochrome P450 2C9: Structural Analysis, Characterization and Comparison.

Cytochromes P450 (CYP) are one of the major xenobiotic metabolizing enzymes with increasing importance in pharmacogenetics. The CYP2C9 enzyme is responsible for the metabolism of a wide range of clinical drugs. More than sixty genetic variations have been identified in CYP2C9 with many demonstrating reduced activity compared to the wild-type (WT) enzyme. The CYP2C9*8 allele is predominantly found in persons of African ancestry and results in altered clearance of several drug substrates of CYP2C9. The X-ray crystal structure of CYP2C9*8, which represents an amino acid variation from arginine to histidine at position 150 (R150H), was solved in complex with losartan. The overall conformation of the CYP2C9*8-losartan complex was similar to the previously solved complex with wild type (WT) protein, but it differs in the occupancy of losartan. One molecule of losartan was bound in the active site and another on the surface in an identical orientation to that observed in the WT complex. However, unlike the WT structure, the losartan in the access channel was not observed in the *8 complex. Furthermore, isothermal titration calorimetry studies illustrated weaker binding of losartan to *8 compared to WT. Interestingly, the CYP2C9*8 interaction with losartan was not as weak as the CYP2C9*3 variant, which showed up to three-fold weaker average dissociation constant compared to the WT. Taken together, the structural and solution characterization yields insights into the similarities and differences of losartan binding to CYP2C9 variants and provides a useful framework for probing the role of amino acid substitution and substrate dependent activity.

Cathelicidin aggravates myocardial ischemia/reperfusion injury via activating TLR4 signaling and P2X7R/NLRP3 inflammasome.

AIMS: The antimicrobial peptide cathelicidin (Camp) has multifunctional immunomodulatory activities. However, its roles in inflammation-related myocardial ischemia/reperfusion (MI/R) injury remain unclear. METHODS AND RESULTS: In this study, adult male C57BL/6 wild-type (WT) mice were subjected to MI/R injury by left anterior descending coronary artery ligation for 45 min followed by 3 or 24 h of reperfusion. An abundant cardiac expression of cathelicidin was observed during ischemia and reperfusion, which was mainly derived from heart-infiltrating neutrophils. Knockout of Camp in mice reduced MI/R-induced myocardial inflammation, infarct size, and circulating cTnI levels (an indicator of heart damage). CRAMP (the mature form of murine cathelicidin) administration of WT mice immediately before MI/R exerted detrimental effects on the reperfused heart. CRAMP exacerbates MI/R injury via a TLR4 and P2X7R/NLRP3 inflammasome-dependent mechanism, since I/R-induced myocardial infarction was reserved by inhibition of TLR4, P2X7R, or NLRP3 inflammasome in CRAMP-treated WT mice. Depletion of neutrophils before MI/R abrogated the amplification of infarct size in CRAMP-treated WT mice. Heart-infiltrating neutrophils were found to be one of major cellular sources of myocardial IL-1ß (a "first line" pro-inflammatory cytokine) at the early stage of MI/R. At this stage, CRAMP administration just before MI/R induced pro-IL-1ß protein expression in heart-infiltrating neutrophils, but not in non-neutrophils. In vitro experiments showed that LL-37 (the mature form of human cathelicidin) treatment promotes the processing and secretion of IL-1ß from human neutrophils via stimulating TLR4 signaling and P2X7R/NLRP3 inflammasome. CONCLUSIONS: Our findings reveal that, at the early stage of MI/R, neutrophil-derived cathelicidin plays an injurious role in the heart. Cathelicidin aggravates MI/R injury by over-activating TLR4 signaling and P2X7R/NLRP3 inflammasome in heart-infiltrating neutrophils, which leads to the excessive secretion of IL-1ß and subsequent inflammatory injury.

Structure of Cytochrome P450 2C9*2 in Complex with Losartan: Insights into the Effect of Genetic Polymorphism.

The human CYP2C9 plays a crucial role in the metabolic clearance of a wide range of clinical therapeutics. The *2 allele is a prevalent genetic variation in CYP2C9 that is found in various populations. A marked reduction of catalytic activity toward many important drug substrates has been demonstrated by CYP2C9*2, which represents an amino acid variation at position 144 from arginine to cysteine. The crystal structure of CYP2C9*2 in complex with an antihypertensive drug losartan was solved using X-ray crystallography at 3.1-Å resolution. The Arg144Cys variation in the *2 complex disrupts the hydrogen-bonding interactions that were observed between the side chain of arginine and neighboring residues in the losartan complex of CYP2C9 and the wild-type (WT) ligand-free structure. The conformation of several secondary structural elements is affected, thereby altering the binding and orientation of drug and important amino acid side chains in the distal active site cavity. The new structure revealed distinct interactions of losartan in the compact active site of CYP2C9*2 and differed in occupancy at the other binding sites previously identified in the WT-losartan complex. Furthermore, the binding studies in solution using losartan illustrated lower activity of the CYP2C9*2 compared with the WT. Together, the findings yield valuable insights into the decreased hydroxylation activity of losartan in patients carrying CYP2C9*2 allele and provide a useful framework to investigate the effect of a single-nucleotide polymorphism that leads to altered metabolism of diverse drug substrates. SIGNIFICANCE STATEMENT: The *2 allele of the human drug-metabolizing enzyme CYP2C9 is found in different populations and results in significantly reduced activity toward various drug substrates. How the CYP2C9*2 variant induces altered drug metabolism is poorly understood given that the Arg144Cys variation is located far away from the active site. This work yield insight into the effect of distal variation using multitude of techniques that include X-ray crystallography, isothermal titration calorimetry, enzymatic characterization, and computational studies.

Rheb (Ras Homolog Enriched in Brain 1) Deficiency in Mature Macrophages Prevents Atherosclerosis by Repressing Macrophage Proliferation, Inflammation, and Lipid Uptake.

OBJECTIVE: Macrophage foam cell formation is an important process in atherosclerotic plaque development. The small GTPase Rheb (Ras homolog enriched in brain 1) regulates endocytic trafficking that is critical for foam cell formation. However, it is unclear whether and how macrophage Rheb regulates atherogenesis, which are the focuses of the current study. Approach and Results: Immunofluorescence study confirmed the colocalization of Rheb in F4/80 and Mac-2 (galectin-3)-labeled lesional macrophages. Western blot and fluorescence-activated cell sorting analysis showed that Rheb expression was significantly increased in atherosclerotic lesions of atherosclerosis-prone (apoE-/- [apolipoprotein E deficient]) mice fed with Western diet. Increased Rheb expression was also observed in oxidized LDL (low-density lipoprotein)-treated macrophages. To investigate the in vivo role of macrophage Rheb, we established mature RhebmKO (macrophage-specific Rheb knockout) mice by crossing the Rheb floxed mice with F4/80-cre mice. Macrophage-specific knockout of Rheb in mice reduced Western diet-induced atherosclerotic lesion by 32%, accompanied with a decrease in macrophage content in plaque. Mechanistically, loss of Rheb in macrophages repressed oxidized LDL-induced lipid uptake, inflammation, and macrophage proliferation. On the contrary, lentivirus-mediated overexpression of Rheb in macrophages increased oxidized LDL-induced lipid uptake and inflammation, and the stimulatory effect of Rheb was suppressed by the mTOR (mammalian target of rapamycin) inhibitor rapamycin or the PKA (protein kinase A) activator forskolin. CONCLUSIONS: Macrophage Rheb plays important role in Western diet-induced atherosclerosis by promoting macrophage proliferation, inflammation, and lipid uptake. Inhibition of expression and function of Rheb in macrophages is beneficial to prevent diet-induced atherosclerosis.

Screening and identification of MicroRNAs expressed in perirenal adipose tissue during rabbit growth.

BACKGROUND: MicroRNAs (miRNAs) regulate adipose tissue development, which are closely related to subcutaneous and intramuscular fat deposition and adipocyte differentiation. As an important economic and agricultural animal, rabbits have low adipose tissue deposition and are an ideal model to study adipose regulation. However, the miRNAs related to fat deposition during the growth and development of rabbits are poorly defined. METHODS: In this study, miRNA-sequencing and bioinformatics analyses were used to profile the miRNAs in rabbit perirenal adipose tissue at 35, 85 and 120 days post-birth. Differentially expressed (DE) miRNAs between different stages were identified by DEseq in R. Target genes of DE miRNAs were predicted by TargetScan and miRanda. To explore the functions of identified miRNAs, Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. RESULTS: Approximately 1.6 GB of data was obtained by miRNA-seq. A total of 987 miRNAs (780 known and 207 newly predicted) and 174 DE miRNAs were identified. The miRNAs ranged from 18 nt to 26 nt. GO enrichment and KEGG pathway analyses revealed that the target genes of the DE miRNAs were mainly involved in zinc ion binding, regulation of cell growth, MAPK signaling pathway, and other adipose hypertrophy-related pathways. Six DE miRNAs were randomly selected, and their expression profiles were validated by q-PCR. CONCLUSIONS: This is the first report of the miRNA profiles of adipose tissue during different growth stages of rabbits. Our data provide a theoretical reference for subsequent studies on rabbit genetics, breeding and the regulatory mechanisms of adipose development.

Deletion of Mtu1 (Trmu) in zebrafish revealed the essential role of tRNA modification in mitochondrial biogenesis and hearing function.

Mtu1(Trmu) is a highly conserved tRNA modifying enzyme responsible for the biosynthesis of τm5s2U at the wobble position of tRNAGln, tRNAGlu and tRNALys. Our previous investigations showed that MTU1 mutation modulated the phenotypic manifestation of deafness-associated mitochondrial 12S rRNA mutation. However, the pathophysiology of MTU1 deficiency remains poorly understood. Using the mtu1 knock-out zebrafish generated by CRISPR/Cas9 system, we demonstrated the abolished 2-thiouridine modification of U34 of mitochondrial tRNALys, tRNAGlu and tRNAGln in the mtu1 knock-out zebrafish. The elimination of this post-transcriptional modification mediated mitochondrial tRNA metabolisms, causing the global decreases in the levels of mitochondrial tRNAs. The aberrant mitochondrial tRNA metabolisms led to the impairment of mitochondrial translation, respiratory deficiencies and reductions of mitochondrial ATP production. These mitochondria dysfunctions caused the defects in hearing organs. Strikingly, mtu1-/- mutant zebrafish displayed the abnormal startle response and swimming behaviors, significant decreases in the sizes of saccular otolith and numbers of hair cells in the auditory and vestibular organs. Furthermore, mtu1-/- mutant zebrafish exhibited the significant reductions in the hair bundle densities in utricle, saccule and lagena. Therefore, our findings may provide new insights into the pathophysiology of deafness, which was manifested by the deficient modifications at wobble position of mitochondrial tRNAs.

Biotransformation of phenolics and metabolites and the change in antioxidant activity in kiwifruit induced by Lactobacillus plantarum fermentation.

BACKGROUND: Changes in antioxidant activity of fruit during fermentation are related to changes in the composition of phenolic acids and flavonoids. In this study, we investigated the effects of Lactobacillus plantarum on the phenolic profile, antioxidant activities, and metabolites of kiwifruit pulp. RESULTS: Lactobacillus plantarum fermentation increased scavenging activity of 1-diphenyl-2-picrylhydrazyl (DPPH) and 2,20-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) radicals. The content of phenolics and flavonoids was increased after fermentation. Correlation analysis demonstrated that the phenolic and flavonoid content was responsible for increasing the scavenging activities of DPPH and ABTS. Lactobacillus plantarum influenced the phenolic profile of the pulp. Protocatechuic and chlorogenic acids were the predominant phenolic acids in fermented kiwifruit pulp. Gallic acid, chlorogenic acid, epicatechin, and catechins were degraded by L. plantarum. The content of 6,7-dihydroxy coumarin and p-coumaric acid, and especially protocatechuic acid, was increased by fermentation. Metabolic differences in lactic acid, fructose, phosphoric acid, gluconolactone, and sugar were evident between non-fermented and fermented kiwifruit. CONCLUSION: Lactobacillus plantarum fermentation increased antioxidant compounds and antioxidant activity in kiwifruit pulp. These results provide the foundation to target the functional benefits of L. plantarum-fermented kiwifruit pulp for further human, animal, and plant health applications. © 2020 Society of Chemical Industry.

A visual sensor array based on an indicator displacement assay for the detection of carboxylic acids.

Carboxylic acids (CAs) have been reported as potential biomarkers of specific diseases or human body odors. A visual sensor array is described here that is based on indicator displacement assays (IDAs). The arrays were prepared by spotting solutions of the following metal complexes: Murexide-Ni(II), murexide-Cu(II), zincon-Zn(II) and xylenol orange-Cu(II), with the capability of discrimination of 15 carboxylic acids (CAs) and the quantitation of pyruvic acid (PA). Clear differences can be observed through distinctive difference maps obtained within 5 min by subtraction of red, green and blue (RGB) values of digital images after and before exposure to analytes. After an analysis of multidimensional data by pattern recognition algorithms including HCA, PCA and LDA, excellent classification specificity, and accuracy of >96% were obtained for all samples. The IDA array exhibited a linear range from 10 to 1500 µM with a theoretical detection limit of 3.5 µM towards PA. Recoveries of real samples varied from 84.8% to 114.3%. As-fabricated IDA sensor array showed an excellent selectivity among other organic interfering substances and a good batch to batch reproducibility, demonstrating its robustness. All these observations suggested that the IDA sensor array is one of the most promising paths for the discrimination of CAs. Graphical abstract Schematic diagram of indicator displacement assay (a), the procedure for acquisition of difference maps (b), and pattern recognitions for CAs (c). The method uses hierarchical cluster analysis (HCA), principal component analysis (PCA) and linear discriminant analysis (LDA).

Sirtuin 3 deficiency aggravates contrast-induced acute kidney injury.

BACKGROUND: Sirtuin 3 (Sirt3) is a key regulator of energy metabolism and oxidative stress. To investigate the role of Sirt3 in contrast-induced acute kidney injury (CIAKI), we established the model both in vivo and in vitro to explore the potential mechanisms. METHODS: In vivo, we established CIAKI models in wild-type (WT) and Sirt3-knockout (Sirt3-KO) mice. Blood urea nitrogen (BUN) and serum creatinine (Scr) were detected by enzyme-linked immunosorbent assay, Glomerular Filtration Rate (GFR) and creatinine clearance were also investigated. We detected the production of reactive oxygen species (ROS) via 2'7'-dichlorodihydro-fluorescein diacetate. The expressions of Sirt3, oxidative stress and apoptosis related markers (MnSOD, Catalase, Acetyl-MnSOD K68, Nox4, Bax, Bcl-2 and Caspase3) were measured and analyzed. In addition, we observed the effect of nicotinamide riboside (NR) on CIAKI in WT and Sirt3-KO mice. In vitro, Sirt3 was knocked out by siRNA transfection method in HK-2 cells. Sirt3, ROS, oxidative stress and apoptosis markers in HK-2 cells were also measured. RESULTS: Our data demonstrated that the levels of Scr and BUN in Sirt3-KO mice were increased while the levels of the GFR and creatinine clearance were decreased in CIAKI mice. In Sirt3-KO or siRNA groups, the activities of MnSOD and Catalase were markedly down-regulated. Also, the expression of Caspase3 were markedly increased and the ratio of Bcl-2/Bax was decreased, while the ROS level was increased in Sirt3 deficiency groups. NR ameliorated CIAKI in WT mice but not in Sirt3-KO mice. CONCLUSION: Our results suggest that Sirt3 deficiency aggravates contrast-induced acute kidney injury. Sirt3 is critical in NR-mediated renoprotection in CIAKI.

Crystal Structure of CYP2B6 in Complex with an Efavirenz Analog.

The over two dozen CYP2B structures of human, rabbit, and woodrat enzymes solved in the last decade have significantly enhanced our understanding of the structure-function relationships of drug metabolizing enzymes. More recently, an important role has emerged for halogen-π interactions in the CYP2B6 active site in substrate selectivity, explaining in part the preference for halogenated ligands as substrates. The mechanism by which such ligands interact with CYP2B enzymes involves conserved phenylalanine side chains, in particular F108, F115, or F297, in the active site, which form π bonds with halogens. To illustrate such halogen-π interactions using drugs that are major substrates of CYP2B6, we present here a crystal structure of CYP2B6 in complex with an analog of the widely used anti-HIV drug efavirenz, which contains a methyl group in place of the carbonyl oxygen. The chlorine of the efavirenz analog forms a π bond with the aromatic ring of F108, whereas the putative metabolism site on the distal end of the molecule is oriented towards the heme iron. The crystal structure showcases how CYP2B6 accommodates this important drug analog of considerable size in the active site by movement of various side chains without substantially increasing the active site volume. Furthermore, the CYP2B6-efavirenz analog complex provides a useful platform to investigate computationally as well as biophysically the effect of genetic polymorphisms on binding of the widely studied efavirenz.

Structural Basis of Single-Nucleotide Polymorphisms in Cytochrome P450 2C9.

Single-nucleotide polymorphisms in drug-metabolizing cytochrome P450 (CYP) enzymes are important contributors to interindividual differences in drug metabolism leading to adverse drug reactions. Despite their extensive characterization and importance in pharmacogenetics of clinical drugs, the structural basis of CYP polymorphisms has remained scant. Here we report the crystal structures of human CYP2C9 and its polymorphic variants, *3 (I359L) and *30 (A477T), with an antihypertensive drug losartan. The structures show distinct interaction and occupation of losartan in the active site, the access channel, and the peripheral binding site. The I359L substitution located far from the active site remarkably altered the residue side chains near the active site and the access channel, whereas the T477 substitution illustrated hydrogen-bonding interaction with the reoriented side chain of Q214. The results yield structural insights into the reduced catalytic activity of the CYP2C9 variants and have important implications for understanding genetic polymorphisms in CYP-mediated drug metabolism.