Coenzyme Q10 deficiency and response to supplementation in pediatric and adolescent migraine

BACKGROUND: Coenzyme Q10 (CoQ10) has been suggested to be effective in the prevention of migraine, and levels can be quantified with standardized reference ranges. OBJECTIVE: This study documents the prevalence of CoQ10 deficiency in migraine headache and examines the potential effectiveness of supplementation. METHODS: We assessed patients attending a tertiary care center with frequent headaches for CoQ10 deficiency. We recommended patients with low CoQ10 levels begin supplementation with CoQ10 as part of their multidisciplinary treatment plan. We assessed response to treatment including correction of CoQ10 deficiency, overall headache improvement, and headache disability. RESULTS: CoQ10 was measured in 1550 patients (mean age 13.3 +/- 3.5, range 3 to 22 years). The mean total CoQ10 level was 0.60 +/- 0.20 microg/mL (range 0.21 to 1.77 microg/mL). Of these patients, 32.9% were below the reference range. Patients with low CoQ10 were recommended to start 1 to 3 mg/kg per day of CoQ10 in liquid gel capsule formulation. In a subset of patients who returned for timely follow-up (mean, 97 days), the total CoQ10 level improved to 1.20 +/- 0.59 microg/mL (P < .0001), while the headache frequency improved from 19.2 +/- 10.0 to 12.5 +/- 10.8 (P < .001) and headache disability assessed with PedMIDAS improved from 47.4 +/- 50.6 to 22.8 +/- 30.6 (P < .001). CONCLUSIONS: Deficiency of CoQ10 may be common in pediatric and adolescent migraine. Determination of deficiency and consequent supplementation may result in clinical improvement. Further analysis involving more scientifically rigorous methodology will be required to confirm this observation.     

Spatial Arrangement of Ribosomal Proteins: Reaction of the Escherichia coli 30S Subunit with bis-Imidoesters

The 30S ribosomal subunit of E. coli was treated with the bifunctional reagent bis-(methyl)suberimidate. Crosslinked ribosomal proteins were identified as bands with increased molecular weight after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. The pattern of crosslinked products was altered when unfolded subunits were used. Free ribosomal protein was not crosslinked. Several of the crosslinked products were cleaved by ammonolysis to form the original monomeric protein constituents. The low yields of the reactions necessitated the use of radioactive proteins and auto-radiographic procedures. The crosslinked proteins were tentatively identified by coelectrophoresis of the radioactive ammonolysis products with carrier 30S protein in sodium dodecyl sulphate, and coelectrophoresis at pH 4.5 in buffers containing urea.     

Molecular cloning of cDNA for Avena phytochrome

We have isolated several cDNA clones for phytochrome, a plant regulatory photoreceptor. A cDNA library was constructed by using etiolated Avena poly(A)⁺ RNA enriched for phytochrome mRNA by size fractionation. Replicate arrays of colonies were differentially screened with cDNA probes made from poly(A)⁺ RNA that had been either enriched in or depleted of phytochrome mRNA. Of the colonies hybridizing preferentially with the enriched probe, several contained plasmids that specifically selected phytochrome mRNA when assayed by hybridization-selection and translation. The largest such plasmid, pAP-2, was used to isolate clones from an Avena genomic library. One of these genomic clones was then used to screen a second cDNA library in an attempt to identify full-length phytochrome clones. The largest of the plasmids thus obtained, pAP-3, contains a 3.4-kilobasepair (kbp) insert, verified to contain phytochrome sequences by hybridization-selection and translation. Sequence analysis of pAP-2 and pAP-3 revealed that the two clones are identical in sequence through a 2.4-kbp region in which they overlap. However, the pAP-2 insert contains, in addition, 1.5 kbp of sequence of unknown origin, the apparent result of a recombination event. Blots of poly(A)⁺ RNA hybridized with ³²P-labeled pAP-2 or pAP-3 show a single mRNA band at 4.2 kilobases. Blot analysis of RNA from dark-grown and from red-irradiated tissue demonstrates that a previously reported light-induced decrease in translatable phytochrome mRNA results from a decrease in physical abundance of this mRNA.     

A protease-mediated switch regulates the growth of magnetosome organelles in Magnetospirillum magneticum

Magnetosomes are lipid-bound organelles that direct the biomineralization of magnetic nanoparticles in magnetotactic bacteria. Magnetosome membranes are not uniform in size and can grow in a biomineralization-dependent manner. However, the underlying mechanisms of magnetosome membrane growth regulation remain unclear. Using cryoelectron tomography, we systematically examined mutants with defects at various stages of magnetosome formation to identify factors involved in controlling membrane growth. We found that a conserved serine protease, MamE, plays a key role in magnetosome membrane growth regulation. When the protease activity of MamE is disrupted, magnetosome membrane growth is restricted, which, in turn, limits the size of the magnetite particles. Consistent with this finding, the upstream regulators of MamE protease activity, MamO and MamM, are also required for magnetosome membrane growth. We then used a combination of candidate and comparative proteomics approaches to identify Mms6 and MamD as two MamE substrates. Mms6 does not appear to participate in magnetosome membrane growth. However, in the absence of MamD, magnetosome membranes grow to a larger size than the wild type. Furthermore, when the cleavage of MamD by MamE protease is blocked, magnetosome membrane growth and biomineralization are severely inhibited, phenocopying the MamE protease-inactive mutant. We therefore propose that the growth of magnetosome membranes is controlled by a protease-mediated switch through processing of MamD. Overall, our work shows that, like many eukaryotic systems, bacteria control the growth and size of biominerals by manipulating the physical properties of intracellular organelles.

Biomineralization is a common phenomenon across the tree of life; one type is a biologically controlled mineral production process that is often initiated within intracellular membrane-bound organelles or vesicle-like structures (1, 2). For instance, matrix vesicles serve as initial sites for mineral formation in the growth plate and most other vertebrate mineralization tissues (3). Vesicles also play a central role in the formation of calcitic spicules in sea urchins (4), extracellular calcitic plates in marine coccolithophores (5), and the silica-based cell walls of diatoms (6). Compartmentalization within a membrane is believed to provide an isolated microenvironment and a template for efficient nucleation, growth, and shaping of minerals.In contrast to the multiple examples of eukaryotic biomineralization noted here, little is known regarding the diversity and dynamics of bacterial biomineralization at the molecular and cellular level. Production of magnetic minerals within magnetosome organelles of magnetotactic bacteria (MTB) stands as one of the best-studied examples of biomineralization in bacteria. MTB are a diverse group of gram-negative bacteria often found near the oxic-anoxic transition zone of aquatic environments (7). Magnetic nanoparticles (magnetite or greigite) mineralized by MTB are generally 35 to 120 nm in length, a size range that yields a single, stable magnetic moment (8). Magnetosomes are typically arranged into one or multiple chains that function as a complete magnetic unit, enabling MTB to navigate along geomagnetic field lines and efficiently find the oxic-anoxic transition zone in a process termed magneto-aerotaxis (9).Biomineralization compartments generally contain a specific cohort of proteins that play critical roles during organelle formation and mineralization. Proteins involved in magnetosome biogenesis are normally encoded from a genomic region called the magnetosome gene island (MAI) (SI Appendix, Fig. S1A). Many magnetosome‐associated membrane (Mam) and magnetic particle membrane‐specific (Mms) proteins are associated with magnetosomes (10–12). The genes encoding the Mam and Mms proteins are organized into four clusters (mamAB, mamGFDC, mms6, and mamXY) in the model Magnetosprillum species and are necessary and sufficient for magnetosome formation (13–17) (Fig. 1A). Analyses of deletion mutants have been used to assign roles for individual genes in one of four distinct stages of magnetosome biogenesis in the model organism Magnetospirillum magneticum AMB-1 (AMB-1): 1) empty membrane invagination (mamI, -L, -Q, and -B), 2) chain alignment (mamK, -J, and -Y), 3) crystal nucleation (mamM, -N, and -O), and 4) crystal maturation (other genes within the four clusters) (13, 14, 18) (Fig. 1A).Open in a separate windowFig. 1.The essential genes and the process of magnetosome production. (A) Schematic depicting the four key magnetosome gene clusters of AMB-1. A total of 10 genes were tested in this study for magnetosome membrane growth regulation: genes involved in crystal initiation (mamM, -N, and -O) are marked in orange, and genes involved in crystal maturation (mamE, -P, -A, -S, -T, -D, and mms6) are marked in blue. Based on previous work, mmsF is known to not be involved in magnetosome membrane growth (8). (B) Model of the biomineralization-dependent magnetosome membrane growth based on Cornejo et al. 2016 (19). OM, outer membrane. IM, inner membrane.The resulting stepwise model outlines a set of processes that are seemingly distinct from one another. However, examination of the dynamics of magnetosome formation has revealed that magnetosome membrane growth is closely linked to the progression of biomineralization. Within a given AMB-1 cell, the magnetosome chain consists of some empty magnetosome membranes (EMMs) as well as the crystal-containing magnetosome membranes (CMMs) that provide the dipole moment necessary for orientation in magnetic fields. Cornejo et al. showed that at steady state, the diameter of the magnetosome lumen ranges from 20 to 80 nm (volume of about 4,189 to 268,083 nm³), yet no EMMs grow beyond 55 nm (volume of about 65,450 nm³) (19). Accordingly, when biomineralization is disrupted by limiting iron availability, only EMMs are produced and their growth stalls at about 55 nm, implying the existence of a checkpoint for membrane growth (Fig. 1B). Upon iron addition, membranes that have initiated biomineralization (CMMs) grow larger than this limit, implying that active biomineralization is needed for further membrane growth (19) leading to a linear relationship between the size of the growing crystals and the surrounding membranes (Fig. 1B). One possible explanation for these observations is that the growing mineral pushes against the membrane and drives its expansion. However, a mutant missing MmsF, a late-stage biomineralization protein, makes small magnetite crystals and still produces membranes as large as the wild-type (WT) parent in a biomineralization-dependent manner (19). These observations imply that magnetosome membrane growth is tightly regulated to create an optimal environment for crystal nucleation, which triggers the second membrane growth stage for crystal maturation (19).The discovery of biomineralization-dependent magnetosome membrane growth provides a lens to examine the function of magnetosome proteins. One hypothesis holds that regulated growth of the magnetosome membrane allows for proper accumulation of iron to high concentrations to initiate nucleation and growth of magnetic particles. Thus, factors known to influence the growth and geometry of magnetite crystals may actually do so by regulating the physical properties of the magnetosome membrane. Here, we explored this possibility by using whole cell cryoelectron tomography (cryo-ET) to directly measure the sizes of magnetosome membranes in a series of mutants with known defects in crystal production. MamE belongs to a highly conserved high temperature requirement A (HtrA) family of trypsin-like serine proteases, whose activity is required for crystal maturation with an unknown mechanism (20). Here, we find that the catalytic activity of MamE plays a central role in the progression of magnetosome membrane growth. MamE proteolytically processes itself and two other biomineralization factors, MamO and MamP (21). MamO is required for MamE protease activation (22), and we find it acts as an upstream regulator of MamE for magnetosome membrane growth. MamP is not involved in magnetosome membrane growth. We also identified MamD, a protein that binds tightly to magnetite and was previously thought to promote crystal maturation (23, 24), as a direct substrate of MamE and showed that MamD is in fact a negative regulator of biomineralization. Our results indicate that MamE activates membrane remodeling by relieving MamD’s inhibition on the size of the magnetosome lumen and demonstrate how spatial restructuring of an organelle can regulate its biochemical output.     

A Mediterranean lifestyle reduces the risk of cardiovascular disease in the “Seguimiento Universidad de Navarra” (SUN) cohort

Background and aimsA healthy lifestyle is essential to prevent cardiovascular disease (CVD). However, beyond dietary habits, there is a scarcity of studies comprehensively assessing the typical traditional Mediterranean lifestyle with a multi-dimensional index. We assessed the association between the Mediterranean lifestyle (measured with the MEDLIFE index including diet, physical activity, and other lifestyle factors) and the incidence of CVD.Methods and resultsThe “Seguimiento Universidad de Navarra” (SUN) project is a prospective, dynamic and multipurpose cohort of Spanish university graduates. We calculated a MEDLIFE score, composed of 28 items on food consumption, dietary habits, physical activity, rest, social habits, and conviviality, for 18,631 participants by assigning 1 point for each typical Mediterranean lifestyle factor achieved, for a theoretically possible final score ranging from 0 to 28 points. During an average follow-up of 11.5 years, 172 CVD cases (myocardial infarction, stroke or cardiovascular death) were observed. An inverse association between the MEDLIFE score and the risk of primary cardiovascular events was observed, with multivariable-adjusted hazard ratio (HR) = 0.50; (95% confidence interval, 0.31–0.81) for the highest MEDLIFE scores (14–23 points) compared to the lowest scores (0–9 points), p (trend) = 0.004.ConclusionA higher level of adherence to the Mediterranean lifestyle was significantly associated with a lower risk of CVD in a Spanish cohort. Public health strategies should promote the Mediterranean lifestyle to preserve cardiovascular health.