Heterogeneity of mitochondrial creatine kinase

The heterogeneity of cardiac sarcomeric mitochondrial creatine kinase (creatine N-phosphotransferase, EC 2.7.3.2, sMi-CK), namely, brain ubiquitous Mi-CK (uMi-CK) and an atypical Mi-CK detected in the serum of a patient with ovarian cancer, was studied by isoelectric focusing. These Mi-CKs were found to be slightly different from each other with respect to their pIs under the examined conditions. The atypical Mi-CK was found to be an atypically oxidized form of uMi-CK. Results suggest that these heterogeneities of Mi-CK are caused by the genotypes, structures, biological functions and metabolism/dissimilation of Mi-CKs in the mitochondria and intravascular circulation.     

Genomic analysis of a migratory divide reveals candidate genes for migration and implicates selective sweeps in generating islands of differentiation

Differential gene flow, reductions in diversity following linked selection and/or features of the genome can structure patterns of genomic differentiation during the process of speciation. Possible sources of reproductive isolation are well studied between coastal and inland subspecies groups of Swainson's thrushes, with differences in seasonal migratory behaviour likely playing a key role in reducing hybrid fitness. We assembled and annotated a draft reference genome for this species and generated whole‐genome shotgun sequence data for populations adjacent to the hybrid zone between these groups. We documented substantial genomewide heterogeneity in relative estimates of genetic differentiation between the groups. Within population diversity was lower in areas of high relative differentiation, supporting a role for selective sweeps in generating this pattern. Absolute genetic differentiation was reduced in these areas, further suggesting that recurrent selective sweeps in the ancestral population and/or between divergent populations following secondary contact likely occurred. Relative genetic differentiation was also higher near centromeres and on the Z chromosome, suggesting that features of the genome also contribute to genomewide heterogeneity. Genes linked to migratory traits were concentrated in islands of differentiation, supporting previous suggestions that seasonal migration is under divergent selection between Swainson's thrushes. Differences in migratory behaviour likely play a central role in the speciation of many taxa; we developed the infrastructure here to permit future investigations into the role several candidate genes play in reducing gene flow between not only Swainson's thrushes but other species as well.     

Peptoids advance multidisciplinary research and undergraduate education in parallel: Sequence effects on conformation and lipid interactions

Peptoids are versatile peptidomimetic molecules with wide-ranging applications from drug discovery to materials science. An understanding of peptoid sequence features that contribute to both their three-dimensional structures and their interactions with lipids will expand functions of peptoids in varied fields. Furthermore, these topics capture the enthusiasm of undergraduate students who prepare and study diverse peptoids in laboratory coursework and/or in faculty led research. Here, we present the synthesis and study of 21 peptoids with varied functionality, including 19 tripeptoids and 2 longer oligomers. We observed differences in fluorescence spectral features for 10 of the tripeptoids that correlated with peptoid flexibility and relative positioning of chromophores. Interactions of representative peptoids with sonicated glycerophospholipid vesicles were also evaluated using fluorescence spectroscopy. We observed evidence of conformational changes effected by lipids for select peptoids. We also summarize our experiences engaging students in peptoid-based projects to advance both research and undergraduate educational objectives in parallel.     

Mutasynthesis - uniting chemistry and genetics for drug discovery

Mutasynthesis couples the power of chemical synthesis with molecular biology to generate derivatives of medicinally valuable, natural products. Recently, this technique has been exploited by Cambridge-based biotech company Biotica Technology Ltd, and their collaborators, to generate promising new variants of the polyketide anti-cancer compounds rapamycin and borrelidin.     

Increased mitochondrial H2O2 production promotes endothelial NF-kappaB activation in aged rat arteries

Previous studies have shown that the aging vascular system undergoes pro-atherogenic phenotypic changes, including increased oxidative stress and a pro-inflammatory shift in endothelial gene expression profile. To elucidate the link between increased oxidative stress and vascular inflammation in aging, we compared the carotid arteries and aortas of young and aged (24 mo old) Fisher 344 rats. In aged vessels there was an increased NF-kappaB activity (assessed by luciferase reporter gene assay and NF-kappaB binding assay), which was attenuated by scavenging H(2)O(2). Aging did not alter the vascular mRNA and protein expression of p65 and p50 subunits of NF-kappaB. In endothelial cells of aged vessels there was an increased production of H(2)O(2) (assessed by 5,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate-acetyl ester fluorescence), which was attenuated by the mitochondrial uncoupler FCCP. In young arteries and cultured endothelial cells, antimycin A plus succinate significantly increased FCCP-sensitive mitochondrial H(2)O(2) generation, which was associated with activation of NF-kappaB. In aged vessels inhibition of NF-kappaB (by pyrrolidenedithiocarbamate, resveratrol) significantly attenuated inflammatory gene expression and inhibited monocyte adhesiveness. Thus increased mitochondrial oxidative stress contributes to endothelial NF-kappaB activation, which contributes to the pro-inflammatory phenotypic alterations in the aged vaculature. Our model predicts that by reducing mitochondrial H(2)O(2) production and/or directly inhibiting NF-kappaB novel anti-aging pharmacological treatments (e.g., calorie restriction mimetics) will exert significant anti-inflammatory and vasoprotective effects.     

Tissue-Specific Apocarotenoid Glycosylation Contributes to Carotenoid Homeostasis in Arabidopsis Leaves

Attaining defined steady-state carotenoid levels requires balancing of the rates governing their synthesis and metabolism. Phytoene formation mediated by phytoene synthase (PSY) is rate limiting in the biosynthesis of carotenoids, whereas carotenoid catabolism involves a multitude of nonenzymatic and enzymatic processes. We investigated carotenoid and apocarotenoid formation in Arabidopsis (Arabidopsis thaliana) in response to enhanced pathway flux upon PSY overexpression. This resulted in a dramatic accumulation of mainly β-carotene in roots and nongreen calli, whereas carotenoids remained unchanged in leaves. We show that, in chloroplasts, surplus PSY was partially soluble, localized in the stroma and, therefore, inactive, whereas the membrane-bound portion mediated a doubling of phytoene synthesis rates. Increased pathway flux was not compensated by enhanced generation of long-chain apocarotenals but resulted in higher levels of C₁₃ apocarotenoid glycosides (AGs). Using mutant lines deficient in carotenoid cleavage dioxygenases (CCDs), we identified CCD4 as being mainly responsible for the majority of AGs formed. Moreover, changed AG patterns in the carotene hydroxylase mutants lutein deficient1 (lut1) and lut5 exhibiting altered leaf carotenoids allowed us to define specific xanthophyll species as precursors for the apocarotenoid aglycons detected. In contrast to leaves, carotenoid hyperaccumulating roots contained higher levels of β-carotene-derived apocarotenals, whereas AGs were absent. These contrasting responses are associated with tissue-specific capacities to synthesize xanthophylls, which thus determine the modes of carotenoid accumulation and apocarotenoid formation.In plants, the synthesis of carotenoids is plastid localized, with the plastid type determining their function (Ruiz-Sola and Rodríguez-Concepción, 2012; Nisar et al., 2015). In nonphotosynthetic chromoplasts, carotenoids and their volatile derivatives attract pollinating insects or zoochoric animals. Here, carotenoids are sequestered in diverse suborganellar structures, which can be tubulous, globulous, membranous, or crystalline (Sitte et al., 1980; Egea et al., 2010). In chloroplasts, carotenoids are present in light-harvesting complex proteins and photosynthetic reaction centers. They extend the light spectrum used for photosynthetic energy transformation and act photoprotectively because of their ability to quench excitation energy from singlet- or triplet-state chlorophylls, thereby decreasing the risk that singlet oxygen forms (Niyogi, 1999; Demmig-Adams and Adams, 2002). Furthermore, the regulated epoxidation and deepoxidation of zeaxanthin in the xanthophyll cycle contribute to the nonphotochemical quenching of energy (Niyogi, 1999; Ballottari et al., 2014). In contrast to these processes, which maintain carotenoid integrity, carotenoids are also capable of chemically quenching singlet oxygen by their own oxidation, which is accompanied by the release of various carotenoid degradation products (Ramel et al., 2012a, 2013).The various functions of carotenoids require their dynamic qualitative and quantitative tuning in response to environmental conditions to attain and maintain adequate steady-state concentrations. These include both the regulation of their synthesis and the formation, release, or disposal of their breakdown products. The synthesis of carotenoids is initiated by the condensation of two molecules of geranylgeranyl diphosphate to form phytoene catalyzed by the enzyme phytoene synthase (PSY), which is considered as the rate-limiting enzyme (von Lintig et al., 1997; Li et al., 2008; Rodríguez-Villalón et al., 2009; Welsch et al., 2010; Zhou et al., 2015). In plants, two desaturases, phytoene desaturase and ζ-carotene desaturase, and two carotene cis-trans-isomerases convert the colorless phytoene into the red-colored all-trans-lycopene (Isaacson et al., 2002; Park et al., 2002; Chen et al., 2010; Yu et al., 2011). Two lycopene cyclases introduce either β- or ε-ionone rings, yielding α-(ε,β-)-carotene and β-(β,β)-carotene. In Arabidopsis (Arabidopsis thaliana), four enzymes hydroxylate carotenes with partially overlapping substrate specificity (Kim et al., 2009). Two nonheme iron-dependent β-carotene hydroxylases (BCH), BCH1 and BCH2, convert β-carotene into zeaxanthin. The second set of hydroxylases, cytochrome P450 (CYP)97A3 and CYP97C1, prefers α-carotene and produces zeinoxanthin and lutein, respectively. Absence of each cytochrome P450 hydroxylase constitutes a distinct phenotype, named lutein deficient5 (lut5) for CYP97A3 deficiency and lut1 for CYP97C1 deficiency, characterized by altered pigment compositions and the accumulation of monohydroxylated intermediates, whereas deficiency in BCH1 and BCH2 does not affect the pigment composition.In green tissues, photooxidative destruction seemingly predominates and consumes carotenoids (Simkin et al., 2003). Moreover, ¹⁴CO₂ pulse-chase experiments with Arabidopsis leaves identified α- and β-carotene as the main targets for photooxidation, whereas xanthophylls were less affected (Beisel et al., 2010). Oxidation assays with β-carotene showed epoxy- and peroxy-derivatives as the main primary products, which however, undergo additional reactions, yielding more stable degradation products that are, in part, the same apocarotenals/ones as those being produced enzymatically (Ramel et al., 2012a, 2013).In Arabidopsis, genes coding for carotenoid cleaving enzymes (carotenoid cleavage dioxygenases [CCDs]) form a small gene family comprising nine members, five of which are attributed to the synthesis of abscisic acid (ABA; nine-cis-epoxycarotenoid cleavage dioxygenases [NCEDs]; AtNCED2, AtNCED3, AtNCED5, AtNCED6, and AtNCED9; Iuchi et al., 2001; Tan et al., 2003), whereas two are committed to strigolactone biosynthesis (CCD7/MORE AXILLARY GROWTH3 [MAX3] and CCD8/MAX4; Alder et al., 2012; Bruno et al., 2014). Orthologs of CCD1 are involved in the generation of volatile apocarotenoids contributing to flower scents and aroma production (e.g. saffron [Crocus sativus; Rubio et al., 2008; Frusciante et al., 2014] and tomato [Solanum lycopersicum; Simkin et al., 2004]), whereas CCD4 enzymes are involved in citrus peel and chrysanthemum (Chrysanthemum morifolium) petal coloration (Ohmiya et al., 2006; Rodrigo et al., 2013). Recent analysis of Arabidopsis mutants revealed a major function of CCD4 in regulating seed carotenoid content with only a minor contribution of CCD1 (Gonzalez-Jorge et al., 2013). Moreover, CCD4 activity was required for the synthesis of an apocarotenoid-derived signaling molecule involved in leaf development and retrograde gene expression (Avendaño-Vázquez et al., 2014).Elevated carotenoid pathway flux caused by PSY overexpression increases carotenoid accumulation in various nongreen tissues, such as tomato fruits, canola (Brassica napus) seeds, cassava (Manihot esculenta) roots, and rice (Oryza sativa) endosperm (Shewmaker et al., 1999; Ye et al., 2000; Fraser et al., 2002; Welsch et al., 2010). Similarly, the constitutive overexpression of PSY in Arabidopsis results in dramatically increased carotenoid amounts accumulating as crystals in nongreen tissues, such as roots and callus, yielding β-carotene as the main product (Maass et al., 2009). However, leaves of the very same plants do not show altered pigment composition, and phytoene or other pathway intermediates are not detected. Similarly, increased levels of active PSY protein achieved though overexpression of the ORANGE protein exclusively affect carotenoid amounts in roots but not in leaves (Zhou et al., 2015). Leaves from constitutively PSY-overexpressing tomato and tobacco (Nicotiana tabacum) plants are also reported to show only slightly increased carotenoid levels compared with the wild-type control (Fray et al., 1995; Busch et al., 2002). These contrasting responses of leaves versus nongreen tissues to elevated pathway flux suggest fundamental differences in the modes of carotenoid formation and/or degradation.In this work, we identified xanthophyll-derived apocarotenoid glycosides (AGs) in Arabidopsis leaves that increase upon higher pathway flux. This suggests that apocarotenoid glycosylation functions as a valve regulating carotenoid steady-state levels in leaves. The analysis of Arabidopsis mutants enabled us to conclude on potential precursor carotenoids and assess the contribution of carotenoid cleavage enzymes on their formation. Moreover, apocarotenoids but not the identified glycosides were increased in carotenoid-hyperaccumulating roots, indicating tissue-specific different modes of carotenoid turnover regulation.     

Transparency of Outcome Reporting and Trial Registration of Randomized Controlled Trials Published in the Journal of Consulting and Clinical Psychology

Background

Confidence that randomized controlled trial (RCT) results accurately reflect intervention effectiveness depends on proper trial conduct and the accuracy and completeness of published trial reports. The Journal of Consulting and Clinical Psychology (JCCP) is the primary trials journal amongst American Psychological Association (APA) journals. The objectives of this study were to review RCTs recently published in JCCP to evaluate (1) adequacy of primary outcome analysis definitions; (2) registration status; and, (3) among registered trials, adequacy of outcome registrations. Additionally, we compared results from JCCP to findings from a recent study of top psychosomatic and behavioral medicine journals.

Methods

Eligible RCTs were published in JCCP in 2013–2014. For each RCT, two investigators independently extracted data on (1) adequacy of outcome analysis definitions in the published report, (2) whether the RCT was registered prior to enrolling patients, and (3) adequacy of outcome registration.

Results

Of 70 RCTs reviewed, 12 (17.1%) adequately defined primary or secondary outcome analyses, whereas 58 (82.3%) had multiple primary outcome analyses without statistical adjustment or undefined outcome analyses. There were 39 (55.7%) registered trials. Only two trials registered prior to patient enrollment with a single primary outcome variable and time point of assessment. However, in one of the two trials, registered and published outcomes were discrepant. No studies were adequately registered as per Standard Protocol Items: Recommendation for Interventional Trials guidelines. Compared to psychosomatic and behavioral medicine journals, the proportion of published trials with adequate outcome analysis declarations was significantly lower in JCCP (17.1% versus 32.9%; p = 0.029). The proportion of registered trials in JCCP (55.7%) was comparable to behavioral medicine journals (52.6%; p = 0.709).

Conclusions

The quality of published outcome analysis definitions and trial registrations in JCCP is suboptimal. Greater attention to proper trial registration and outcome analysis definition in published reports is needed.