Stabilization of carbon through co-addition of water treatment residuals with anaerobic digested sludge in a coarse textured soil.

Coarse textured soils have low potential to store carbon (C) due to lack of mineral oxides and have low clay content to protect C from biodegradation and leaching. This study evaluated the potential of stabilizing C by adding metal oxyhydroxide-rich water treatment residuals (WTRs) to an aeolian pure sand (<5% clay) topsoil amended with anaerobic digestate (AD) sludge. The AD sludge was applied at 5% (w/w) with aluminum based WTR (Al-WTR) and iron based WTR (Fe-WTR) co-applied at 1:1 and 2:1 WTR:AD (w/w) ratios and incubated at room temperature for 132 days. The cumulative mineralized C was normalized to the total organic C of the treatments. Co-addition with Al-WTR showed to be more effective in stabilizing C through decreased cumulative mineralized C by 48% and 57% in 1Al-WTR:1AD and 2Al-WTR:1AD, respectively, compared to AD sludge sole amendment. Co-application with Al-WTR also decreased permanganate oxidizable C by 37% and dissolved organic C by 51%. Co-application with Fe-WTR did not decrease the concentration of these labile C pools to the same extent, possibly due to the selective use of Fe-WTRs to treat organic-rich raw water. This makes it less effective in stabilizing C in a pure sand relative to Al-WTR due to chemical instability of the Fe-organic complexes. The Al-WTR provides a promising co-amendment to increase C sequestration in pure sands when co-applied with biosolids. The co-amendment approach will not only facilitate C sequestration but also contributes to waste management, aligning to the objectives of a circular economy.

A dedicated flavin-dependent monooxygenase catalyzes the hydroxylation of demethoxyubiquinone into ubiquinone (coenzyme Q) in Arabidopsis.

Ubiquinone (Coenzyme Q) is a vital respiratory cofactor and liposoluble antioxidant. In plants, it is not known how the C-6 hydroxylation of demethoxyubiquinone, the penultimate step in ubiquinone biosynthesis, is catalyzed. The combination of cross-species gene network modeling along with mining of embryo-defective mutant databases of Arabidopsis thaliana identified the embryo lethal locus EMB2421 (At1g24340) as a top candidate for the missing plant demethoxyubiquinone hydroxylase. In marked contrast with prototypical eukaryotic demethoxyubiquinone hydroxylases, the catalytic mechanism of which depends on a carboxylate-bridged di-iron domain, At1g24340 is homologous to FAD-dependent oxidoreductases that instead use NAD(P)H as an electron donor. Complementation assays in Saccharomyces cerevisiae and Escherichia coli demonstrated that At1g24340 encodes a functional demethoxyubiquinone hydroxylase and that the enzyme displays strict specificity for the C-6 position of the benzoquinone ring. Laser-scanning confocal microscopy also showed that GFP-tagged At1g24340 is targeted to mitochondria. Silencing of At1g24340 resulted in 40 to 74% decrease in ubiquinone content and de novo ubiquinone biosynthesis. Consistent with the role of At1g24340 as a benzenoid ring modification enzyme, this metabolic blockage could not be bypassed by supplementation with 4-hydroxybenzoate, the immediate precursor of ubiquinone's ring. Unlike in yeast, in Arabidopsis overexpression of demethoxyubiquinone hydroxylase did not boost ubiquinone content. Phylogenetic reconstructions indicated that plant demethoxyubiquinone hydroxylase is most closely related to prokaryotic monooxygenases that act on halogenated aromatics and likely descends from an event of horizontal gene transfer between a green alga and a bacterium.

COQ11 deletion mitigates respiratory deficiency caused by mutations in the gene encoding the coenzyme Q chaperone protein Coq10.

Coenzyme Q (Q n ) is a vital lipid component of the electron transport chain that functions in cellular energy metabolism and as a membrane antioxidant. In the yeast Saccharomyces cerevisiae, coq1-coq9 deletion mutants are respiratory-incompetent, sensitive to lipid peroxidation stress, and unable to synthesize Q6 The yeast coq10 deletion mutant is also respiratory-deficient and sensitive to lipid peroxidation, yet it continues to produce Q6 at an impaired rate. Thus, Coq10 is required for the function of Q6 in respiration and as an antioxidant and is believed to chaperone Q6 from its site of synthesis to the respiratory complexes. In several fungi, Coq10 is encoded as a fusion polypeptide with Coq11, a recently identified protein of unknown function required for efficient Q6 biosynthesis. Because "fused" proteins are often involved in similar biochemical pathways, here we examined the putative functional relationship between Coq10 and Coq11 in yeast. We used plate growth and Seahorse assays and LC-MS/MS analysis to show that COQ11 deletion rescues respiratory deficiency, sensitivity to lipid peroxidation, and decreased Q6 biosynthesis of the coq10Δ mutant. Additionally, immunoblotting indicated that yeast coq11Δ mutants accumulate increased amounts of certain Coq polypeptides and display a stabilized CoQ synthome. These effects suggest that Coq11 modulates Q6 biosynthesis and that its absence increases mitochondrial Q6 content in the coq10Δcoq11Δ double mutant. This augmented mitochondrial Q6 content counteracts the respiratory deficiency and lipid peroxidation sensitivity phenotypes of the coq10Δ mutant. This study further clarifies the intricate connection between Q6 biosynthesis, trafficking, and function in mitochondrial metabolism.

ADCK4 Deficiency Destabilizes the Coenzyme Q Complex, Which Is Rescued by 2,4-Dihydroxybenzoic Acid Treatment.

BACKGROUND: Mutations in ADCK4 (aarF domain containing kinase 4) generally manifest as steroid-resistant nephrotic syndrome and induce coenzyme Q10 (CoQ10) deficiency. However, the molecular mechanisms underlying steroid-resistant nephrotic syndrome resulting from ADCK4 mutations are not well understood, largely because the function of ADCK4 remains unknown. METHODS: To elucidate the ADCK4's function in podocytes, we generated a podocyte-specific, Adck4-knockout mouse model and a human podocyte cell line featuring knockout of ADCK4. These knockout mice and podocytes were then treated with 2,4-dihydroxybenzoic acid (2,4-diHB), a CoQ10 precursor analogue, or with a vehicle only. We also performed proteomic mass spectrometry analysis to further elucidate ADCK4's function. RESULTS: Absence of Adck4 in mouse podocytes caused FSGS and albuminuria, recapitulating features of nephrotic syndrome caused by ADCK4 mutations. In vitro studies revealed that ADCK4-knockout podocytes had significantly reduced CoQ10 concentration, respiratory chain activity, and mitochondrial potential, and subsequently displayed an increase in the number of dysmorphic mitochondria. However, treatment of 3-month-old knockout mice or ADCK4-knockout cells with 2,4-diHB prevented the development of renal dysfunction and reversed mitochondrial dysfunction in podocytes. Moreover, ADCK4 interacted with mitochondrial proteins such as COQ5, as well as cytoplasmic proteins such as myosin and heat shock proteins. Thus, ADCK4 knockout decreased the COQ complex level, but overexpression of ADCK4 in ADCK4-knockout podocytes transfected with wild-type ADCK4 rescued the COQ5 level. CONCLUSIONS: Our study shows that ADCK4 is required for CoQ10 biosynthesis and mitochondrial function in podocytes, and suggests that ADCK4 in podocytes stabilizes proteins in complex Q in podocytes. Our study also suggests a potential treatment strategy for nephrotic syndrome resulting from ADCK4 mutations.

Bioaccumulation of Cu, Fe, Mn and Zn in native Brachystegia longifolia naturally growing in a copper mining environment of Mufulira, Zambia.

Heavy metal contamination in the soil and the subsequent accumulation in Brachystegia longifolia were investigated as a function of the wind direction and distance from a copper mine in Mufulira, Zambia. Soil and leaves of B. longifolia were collected along transects up to 12 km downwind and 19 km upwind. The total concentration of trace elements in the soil and leaves was determined through pXRF. Plant-available Cu, Fe, Mn, and Zn were extracted in a Mehlich III solution and analyzed using ICP-AES. The degree of soil contamination illustrates that Cu and Fe from the copper mine strongly pollute Mufulira and the surrounding forests. Bioavailable Cu, Fe, Mn, and Zn reduced with increasing distance from the mine. An average of 296 mg/kg Cu, 2337 mg/kg Fe, 1101 mg/kg Mn, and 109 mg/kg Zn were recorded in leaves at the most polluted site. Similarly, 55.21 mg/kg Cu, 516.4 mg/kg Fe, 3196 mg/kg Mn, and 154 mg/kg Zn were recorded at an unpolluted site 19 km upwind. The concentration of Cu and Fe reduced significantly with increasing distance, while Mn and Zn increased significantly. It was further established that B. longifolia leaves accumulated Mn (× 38) and Zn (× 15) more than their respective total concentration in the soil. The concentrations of Cu and Fe found in leaves near the mine, as well as the Mn concentration in leaves across the study sites, could be stressful for B. longifolia tree growth.

The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit ß is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit ß and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

Treatment with 2,4-Dihydroxybenzoic Acid Prevents FSGS Progression and Renal Fibrosis in Podocyte-Specific Coq6 Knockout Mice.

BACKGROUND: Although studies have identified >55 genes as causing steroid-resistant nephrotic syndrome (SRNS) and localized its pathogenesis to glomerular podocytes, the disease mechanisms of SRNS remain largely enigmatic. We recently reported that individuals with mutations in COQ6, a coenzyme Q (also called CoQ10, CoQ, or ubiquinone) biosynthesis pathway enzyme, develop SRNS with sensorineural deafness, and demonstrated the beneficial effect of CoQ for maintenace of kidney function. METHODS: To study COQ6 function in podocytes, we generated a podocyte-specific Coq6 knockout mouse (Coq6podKO ) model and a transient siRNA-based COQ6 knockdown in a human podocyte cell line. Mice were monitored for development of proteinuria and assessed for development of glomerular sclerosis. Using a podocyte migration assay, we compared motility in COQ6 knockdown podocytes and control podocytes. We also randomly assigned 5-month-old Coq6podKO mice and controls to receive no treatment or 2,4-dihydroxybenzoic acid (2,4-diHB), an analog of a CoQ precursor molecule that is classified as a food additive by health authorities in Europe and the United States. RESULTS: Abrogation of Coq6 in mouse podocytes caused FSGS and proteinuria (>46-fold increases in albuminuria). In vitro studies revealed an impaired podocyte migration rate in COQ6 knockdown human podocytes. Treating Coq6podKO mice or cells with 2,4-diHB prevented renal dysfunction and reversed podocyte migration rate impairment. Survival of Coq6podKO mice given 2,4diHB was comparable to that of control mice and significantly higher than that of untreated Coq6podKO mice, half of which died by 10 months of age. CONCLUSIONS: These findings reveal a potential novel treatment strategy for those cases of human nephrotic syndrome that are caused by a primary dysfunction in the CoQ10 biosynthesis pathway.

Metabolism of the Flavonol Kaempferol in Kidney Cells Liberates the B-ring to Enter Coenzyme Q Biosynthesis.

Coenzyme Q (CoQ) is an essential component of the mitochondrial electron transport chain and an important antioxidant present in all cellular membranes. CoQ deficiencies are frequent in aging and in age-related diseases, and current treatments are limited to CoQ supplementation. Strategies that rely on CoQ supplementation suffer from poor uptake and trafficking of this very hydrophobic molecule. In a previous study, the dietary flavonol kaempferol was reported to serve as a CoQ ring precursor and to increase the CoQ content in kidney cells, but neither the part of the molecule entering CoQ biosynthesis nor the mechanism were described. In this study, kaempferol labeled specifically in the B-ring was isolated from Arabidopsis plants. Kidney cells treated with this compound incorporated the B-ring of kaempferol into newly synthesized CoQ, suggesting that the B-ring is metabolized via a mechanism described in plant cells. Kaempferol is a natural flavonoid present in fruits and vegetables and possesses antioxidant, anticancer, and anti-inflammatory therapeutic properties. A better understanding of the role of kaempferol as a CoQ ring precursor makes this bioactive compound a potential candidate for the design of interventions aiming to increase endogenous CoQ biosynthesis and may improve CoQ deficient phenotypes in aging and disease.

Human COQ10A and COQ10B are distinct lipid-binding START domain proteins required for coenzyme Q function.

Coenzyme Q (CoQ or ubiquinone) serves as an essential redox-active lipid in respiratory electron and proton transport during cellular energy metabolism. CoQ also functions as a membrane-localized antioxidant protecting cells against lipid peroxidation. CoQ deficiency is associated with multiple human diseases; CoQ10 supplementation in particular has noted cardioprotective benefits. In Saccharomyces cerevisiae, Coq10, a putative START domain protein, is believed to chaperone CoQ to sites where it functions. Yeast coq10 deletion mutants (coq10Δ) synthesize CoQ inefficiently during log phase growth and are respiratory defective and sensitive to oxidative stress. Humans have two orthologs of yeast COQ10, COQ10A and COQ10B Here, we tested the human co-orthologs for their ability to rescue the yeast mutant. We showed that expression of either human ortholog, COQ10A or COQ10B, rescues yeast coq10Δ mutant phenotypes, restoring the function of respiratory-dependent growth on a nonfermentable carbon source and sensitivity to oxidative stress induced by treatment with PUFAs. These effects indicate a strong functional conservation of Coq10 across different organisms. However, neither COQ10A nor COQ10B restored CoQ biosynthesis when expressed in the yeast coq10Δ mutant. The involvement of yeast Coq10 in CoQ biosynthesis may rely on its interactions with another protein, possibly Coq11, which is not found in humans. Coexpression analyses of yeast COQ10 and human COQ10A and COQ10B provide additional insights to functions of these START domain proteins and their potential roles in other biologic pathways.

Treatment Emergent Violence To Self And Others; A Literature Review of Neuropsychiatric Adverse Reactions For Antidepressant And Neuroleptic Psychiatric Drugs And General Medications.

OBJECTIVE: This paper reviews the literature linking physical violence, directed towards self or others, to serotonergic and dopaminergic psychiatric drugs and general medications. DESIGN/METHODOLOGY/APPROACH: Data about side effects, pharmacogenetics and homeostasis are obtained from articles, electronic Medicines Compendium, DSM-IV-TR, British National Formulary (BNF) and academic books. Statistics have been obtained from articles, The National Confidential Inquiry into Suicide and Homicide by People with Mental Illness, Centre for Mental Health and Risk, Manchester, Mental Health Equalities, National Mental Health Development Unit and the NHS Health and Social Care Information Centre. Classification for neurotoxic conditions and mental illness are obtained from the DSM-IV-TR, DSM-V and ICD-10. FINDINGS: Psychiatric drugs and some general medications have effects that are not always the ones intended. Reactions to different drugs and drug-drug combinations are governed by individual metabolising rates. Phase 1 metabolism takes place via the cytochrome P450 enzymes with 57 human genes identified that are genetically variable i.e. polymorphic. The population are coded as poor, extensive (known as normal), intermediate or ultra rapid metabolisers. Variations in the serotonin transporter gene (5-HTTLPR) and serotonin receptors (5-HT) influence the outcome of serotonergic medications. It is established genetic polymorphisms in the CYP450 and serotoninergic metabolising system cause higher drug blood levels which are associated with neuropsychiatric adverse drug reactions (ADRs), such as akathisia. If not recognised, akathisia, which often precedes violence, suicidality, homicide, mania and psychosis, may be mistaken for new or emergent mental illness and treated with further ineffective, counter-productive psychiatric drugs. RESEARCH LIMITATIONS/IMPLICATIONS: The absence of pharmaceutical data for CYP450 diminishing, null/non- functioning or multiple polymorphisms and variations in the 5-HTTLPR and 5-HT, linking general medications and psychiatric drugs with neuropsychiatric behavioural reactions is notable. There is limited information linking psychiatric drug disruption of homeostasis and neurotransmitters with violence. These issues indicate a need for greater pharmaceutical transparency and further research into the role of CYP450, 5-HTTLPR and 5-HT polymorphism associated neuropsychiatric ADRs for all psychiatric drugs and serotonergic general medications. PRACTICAL IMPLICATIONS: Safer prescribing is important and could be achieved by individual genotype testing, which would identify persons with genetic polymorphisms, who are unable to metabolise drugs. Prevention of violence would enhance peoples' well being, ground floor practitioner and public safety. CONCLUSION: This paper is the first review that implicates certain drugs as a cause of violence due to pharmacogentic polymorphisms and neurotransmitter disruption.

Chromatin-remodeling SWI/SNF complex regulates coenzyme Q6 synthesis and a metabolic shift to respiration in yeast.

Despite its relatively streamlined genome, there are many important examples of regulated RNA splicing in Saccharomyces cerevisiae Here, we report a role for the chromatin remodeler SWI/SNF in respiration, partially via the regulation of splicing. We find that a nutrient-dependent decrease in Snf2 leads to an increase in splicing of the PTC7 transcript. The spliced PTC7 transcript encodes a mitochondrial phosphatase regulator of biosynthesis of coenzyme Q6 (ubiquinone or CoQ6) and a mitochondrial redox-active lipid essential for electron and proton transport in respiration. Increased splicing of PTC7 increases CoQ6 levels. The increase in PTC7 splicing occurs at least in part due to down-regulation of ribosomal protein gene expression, leading to the redistribution of spliceosomes from this abundant class of intron-containing RNAs to otherwise poorly spliced transcripts. In contrast, a protein encoded by the nonspliced isoform of PTC7 represses CoQ6 biosynthesis. Taken together, these findings uncover a link between Snf2 expression and the splicing of PTC7 and establish a previously unknown role for the SWI/SNF complex in the transition of yeast cells from fermentative to respiratory modes of metabolism.

Genetic identification of SNP markers linked to a new grape phylloxera resistant locus in Vitis cinerea for marker-assisted selection.

BACKGROUND: Grape phylloxera (Daktulosphaira vitifoliae Fitch) is a major insect pest that negatively impacts commercial grapevine performance worldwide. Consequently, the use of phylloxera resistant rootstocks is an essential component of vineyard management. However, the majority of commercially available rootstocks used in viticulture production provide limited levels of grape phylloxera resistance, in part due to the adaptation of phylloxera biotypes to different Vitis species. Therefore, there is pressing need to develop new rootstocks better adapted to specific grape growing regions with complete resistance to grape phylloxera biotypes. RESULTS: Grapevine rootstock breeding material, including an accession of Vitis cinerea and V. aestivalis, DRX55 ([M. rotundifolia x V. vinifera] x open pollinated) and MS27-31 (M. rotundifolia specific hybrid), provided complete resistance to grape phylloxera in potted plant assays. To map the genetic factor(s) of grape phylloxera resistance, a F1 V. cinerea x V. vinifera Riesling population was screened for resistance. Heritability analysis indicates that the V. cinerea accession contained a single allele referred as RESISTANCE TO DAKTULOSPHAIRA VITIFOLIAE 2 (RDV2) that confers grape phylloxera resistance. Using genetic maps constructed with pseudo-testcross markers for V. cinerea and Riesling, a single phylloxera resistance locus was identified in V. cinerea. After validating SNPs at the RDV2 locus, interval and linkage mapping showed that grape phylloxera resistance mapped to linkage group 14 at position 16.7 cM. CONCLUSION: The mapping of RDV2 and the validation of markers linked to grape phylloxera resistance provides the basis to breed new rootstocks via marker-assisted selection that improve vineyard performance.

Underutilization of Aldosterone Antagonists in Heart Failure.

Background: Treatment with an aldosterone antagonist (AA) has been shown in multiple trials to reduce heart failure (HF)-related morbidity, mortality, and hospital readmission. American College of Cardiology Foundation (ACCF) and American Heart Association (AHA) treatment guidelines recommend the use of an AA in all HF patients with an ejection fraction ≤35% and no known contraindication. Several studies have documented underuse of AA. Objectives: To determine the proportion of patients who received AA therapy consistent with the ACCF/AHA guidelines. Secondary objectives included determining the proportion of patients who received an AA inconsistent with guidelines and 30- and 90-day readmission rates. Methods: A retrospective chart review was conducted of patients admitted to an inner city academic medical center with a diagnosis of HF between August 16, 2011, and June 5, 2013. Results: A total of 346 HF admissions (87.6% African American) were evaluated. Use of an AA at discharge was consistent with guidelines in 31% of patients. A total of 121 patients (35%) were discharged on an AA. Among the remaining 225 patients who were not discharged on an AA, 170 (75.6%) had no contraindication to therapy. Sixty-one patients were readmitted within 30 days, and a total of 108 patients were readmitted within 90 days. There were no significant differences in readmission rates between patients who were discharged on AA therapy and those who were not. Conclusion: AAs are still underutilized in the treatment of HF.

Identification of Coq11, a new coenzyme Q biosynthetic protein in the CoQ-synthome in Saccharomyces cerevisiae.

Coenzyme Q (Q or ubiquinone) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail and is required for mitochondrial electron transport. In the yeast Saccharomyces cerevisiae, Q is synthesized by the products of 11 known genes, COQ1-COQ9, YAH1, and ARH1. The function of some of the Coq proteins remains unknown, and several steps in the Q biosynthetic pathway are not fully characterized. Several of the Coq proteins are associated in a macromolecular complex on the matrix face of the inner mitochondrial membrane, and this complex is required for efficient Q synthesis. Here, we further characterize this complex via immunoblotting and proteomic analysis of tandem affinity-purified tagged Coq proteins. We show that Coq8, a putative kinase required for the stability of the Q biosynthetic complex, is associated with a Coq6-containing complex. Additionally Q6 and late stage Q biosynthetic intermediates were also found to co-purify with the complex. A mitochondrial protein of unknown function, encoded by the YLR290C open reading frame, is also identified as a constituent of the complex and is shown to be required for efficient de novo Q biosynthesis. Given its effect on Q synthesis and its association with the biosynthetic complex, we propose that the open reading frame YLR290C be designated COQ11.

Yeast Coq9 controls deamination of coenzyme Q intermediates that derive from para-aminobenzoic acid.

Coq9 is a polypeptide subunit in a mitochondrial multi-subunit complex, termed the CoQ-synthome, required for biosynthesis of coenzyme Q (ubiquinone or Q). Deletion of COQ9 results in dissociation of the CoQ-synthome, but over-expression of Coq8 putative kinase stabilizes the CoQ-synthome in the coq9 null mutant and leads to the accumulation of two nitrogen-containing Q intermediates, imino-demethoxy-Q6 (IDMQ6) and 3-hexaprenyl-4-aminophenol (4-AP) when para-aminobenzoic acid (pABA) is provided as a ring precursor. To investigate whether Coq9 is responsible for deamination steps in Q biosynthesis, we utilized the yeast coq5-5 point mutant. The yeast coq5-5 point mutant is defective in the C-methyltransferase step of Q biosynthesis but retains normal steady-state levels of the Coq5 polypeptide. Here, we show that when high amounts of 13C6-pABA are provided, the coq5-5 mutant accumulates both 13C6-imino-demethyl-demethoxy-Q6 (13C6-IDDMQ6) and 13C6-demethyl-demethoxy-Q6 (13C6-DDMQ6). Deletion of COQ9 in the yeast coq5-5 mutant along with Coq8 over-expression and 13C6- pABA labeling leads to the absence of 13C6-DDMQ6, and the nitrogen-containing intermediates 13C6-4-AP and 13C6-IDDMQ6 persist. We describe a coq9 temperature-sensitive mutant and show that at the non-permissive temperature, steady-state polypeptide levels of Coq9-ts19 increased, while Coq4, Coq5, Coq6, and Coq7 decreased. The coq9-ts19 mutant had decreased Q6 content and increased levels of nitrogen-containing intermediates. These findings identify Coq9 as a multi-functional protein that is required for the function of Coq6 and Coq7 hydroxylases, for removal of the nitrogen substituent from pABA-derived Q intermediates, and is an essential component of the CoQ synthome.

Resveratrol and para-coumarate serve as ring precursors for coenzyme Q biosynthesis.

Coenzyme Q (Q or ubiquinone) is a redox-active polyisoprenylated benzoquinone lipid essential for electron and proton transport in the mitochondrial respiratory chain. The aromatic ring 4-hydroxybenzoic acid (4HB) is commonly depicted as the sole aromatic ring precursor in Q biosynthesis despite the recent finding that para-aminobenzoic acid (pABA) also serves as a ring precursor in Saccharomyces cerevisiae Q biosynthesis. In this study, we employed aromatic (13)C6-ring-labeled compounds including (13)C6-4HB, (13)C6-pABA, (13)C6-resveratrol, and (13)C6-coumarate to investigate the role of these small molecules as aromatic ring precursors in Q biosynthesis in Escherichia coli, S. cerevisiae, and human and mouse cells. In contrast to S. cerevisiae, neither E. coli nor the mammalian cells tested were able to form (13)C6-Q when cultured in the presence of (13)C6-pABA. However, E. coli cells treated with (13)C6-pABA generated (13)C6-ring-labeled forms of 3-octaprenyl-4-aminobenzoic acid, 2-octaprenyl-aniline, and 3-octaprenyl-2-aminophenol, suggesting UbiA, UbiD, UbiX, and UbiI are capable of using pABA or pABA-derived intermediates as substrates. E. coli, S. cerevisiae, and human and mouse cells cultured in the presence of (13)C6-resveratrol or (13)C6-coumarate were able to synthesize (13)C6-Q. Future evaluation of the physiological and pharmacological responses to dietary polyphenols should consider their metabolism to Q.

Coenzyme Q supplementation or over-expression of the yeast Coq8 putative kinase stabilizes multi-subunit Coq polypeptide complexes in yeast coq null mutants.

Coenzyme Q biosynthesis in yeast requires a multi-subunit Coq polypeptide complex. Deletion of any one of the COQ genes leads to respiratory deficiency and decreased levels of the Coq4, Coq6, Coq7, and Coq9 polypeptides, suggesting that their association in a high molecular mass complex is required for stability. Over-expression of the putative Coq8 kinase in certain coq null mutants restores steady-state levels of the sensitive Coq polypeptides and promotes the synthesis of late-stage Q-intermediates. Here we show that over-expression of Coq8 in yeast coq null mutants profoundly affects the association of several of the Coq polypeptides in high molecular mass complexes, as assayed by separation of digitonin extracts of mitochondria by two-dimensional blue-native/SDS PAGE. The Coq4 polypeptide persists at high molecular mass with over-expression of Coq8 in coq3, coq5, coq6, coq7, coq9, and coq10 mutants, indicating that Coq4 is a central organizer of the Coq complex. Supplementation with exogenous Q6 increased the steady-state levels of Coq4, Coq7, and Coq9, and several other mitochondrial polypeptides in select coq null mutants, and also promoted the formation of late-stage Q-intermediates. Q supplementation may stabilize this complex by interacting with one or more of the Coq polypeptides. The stabilizing effects of exogenously added Q6 or over-expression of Coq8 depend on Coq1 and Coq2 production of a polyisoprenyl intermediate. Based on the observed interdependence of the Coq polypeptides, the effect of exogenous Q6, and the requirement for an endogenously produced polyisoprenyl intermediate, we propose a new model for the Q-biosynthetic complex, termed the CoQ-synthome.

Molecular characterization of the human COQ5 C-methyltransferase in coenzyme Q10 biosynthesis.

Coq5 catalyzes the only C-methylation involved in the biosynthesis of coenzyme Q (Q or ubiquinone) in humans and yeast Saccharomyces cerevisiae. As one of eleven polypeptides required for Q production in yeast, Coq5 has also been shown to assemble with the multi-subunit complex termed the CoQ-synthome. In humans, mutations in several COQ genes cause primary Q deficiency, and a decrease in Q biosynthesis is associated with mitochondrial, cardiovascular, kidney and neurodegenerative diseases. In this study, we characterize the human COQ5 polypeptide and examine its complementation of yeast coq5 point and null mutants. We show that human COQ5 RNA is expressed in all tissues and that the COQ5 polypeptide is associated with the mitochondrial inner membrane on the matrix side. Previous work in yeast has shown that point mutations within or adjacent to conserved COQ5 methyltransferase motifs result in a loss of Coq5 function but not Coq5 steady state levels. Here, we show that stabilization of the CoQ-synthome within coq5 point mutants or by over-expression of COQ8 in coq5 null mutants permits the human COQ5 homolog to partially restore coq5 mutant growth on respiratory media and Q6 content. Immunoblotting against the human COQ5 polypeptide in isolated yeast mitochondria shows that the human Coq5 polypeptide migrates in two-dimensional blue-native/SDS-PAGE at the same high molecular mass as other yeast Coq proteins. The results presented suggest that human and Escherichia coli Coq5 homologs expressed in yeast retain C-methyltransferase activity but are capable of rescuing the coq5 yeast mutants only when the CoQ-synthome is assembled.

Evaluation of the physician quality improvement initiative: the expected and unexpected opportunities.

BACKGROUND: The Physician Quality Improvement Initiative (PQII) uses a well-established multi-source feedback program, and incorporates an additional facilitated feedback review with their department chief. The purpose of this mixed methods study was to examine the value of the PQII by eliciting feedback from various stakeholders. METHODS: All participants and department chiefs (n = 45) were invited to provide feedback on the project implementation and outcomes via survey and/or an interview. The survey consisted of 12 questions focused on the value of the PQII, it's influence on practice and the promotion of quality improvement and accountability. RESULTS: A total of 5 chiefs and 12 physician participants completed semi structured interviews. Participants found the PQII process, report and review session helpful, self-affirming or an opportunity for self-reflection, and an opportunity to engage their leaders about their practice. Chiefs indicated the sessions strengthened their understanding, ability to communicate and engage physicians about their practice, best practices, quality improvement and accountability. Thirty participants (66.7 %) completed the survey; of the responders 75.9, 89.7, 86.7 % found patient, co-worker, and physician colleague feedback valuable, respectively. A total of 67.9 % valued their facilitated review with their chief and 55.2 % indicated they were contemplating change due to their feedback. Participants believed the PQII promoted quality improvement (27/30, 90.0 %), and accountability (28/30, 93.3 %). CONCLUSIONS: The PQII provides an opportunity for physician development, affirmation and reflection, but also a structure to further departmental quality improvement, best practices, and finally, an opportunity to enhance communication, accountability and relationships between the organization, department chiefs and their staff.