The Domain-Specific and Temperature-Dependent Protein Misfolding Phenotype of Variant Medium-Chain acyl-CoA Dehydrogenase

The implementation of expanded newborn screening programs reduced mortality and morbidity in medium-chain acyl-CoA dehydrogenase deficiency (MCADD) caused by mutations in the ACADM gene. However, the disease is still potentially fatal. Missense induced MCADD is a protein misfolding disease with a molecular loss-of-function phenotype. Here we established a comprehensive experimental setup to analyze the structural consequences of eight ACADM missense mutations (p.Ala52Val, p.Tyr67His, p.Tyr158His, p.Arg206Cys, p.Asp266Gly, p.Lys329Glu, p.Arg334Lys, p.Arg413Ser) identified after newborn screening and linked the corresponding protein misfolding phenotype to the site of side-chain replacement with respect to the domain. With fever being the crucial risk factor for metabolic decompensation of patients with MCADD, special emphasis was put on the analysis of structural and functional derangements related to thermal stress. Based on protein conformation, thermal stability and kinetic stability, the molecular phenotype in MCADD depends on the structural region that is affected by missense-induced conformational changes with the central β-domain being particularly prone to structural derangement and destabilization. Since systematic classification of conformational derangements induced by ACADM mutations may be a helpful tool in assessing the clinical risk of patients, we scored the misfolding phenotype of the variants in comparison to p.Lys329Glu (K304E), the classical severe mutation, and p.Tyr67His (Y42H), discussed to be mild. Experiments assessing the impact of thermal stress revealed that mutations in the ACADM gene lower the temperature threshold at which MCAD loss-of-function occurs. Consequently, increased temperature as it occurs during intercurrent infections, significantly increases the risk of further conformational derangement and loss of function of the MCAD enzyme explaining the life-threatening clinical courses observed during fever episodes. Early and aggressive antipyretic treatment thus may be life-saving in patients suffering from MCADD.     

Super-genotype: global monoclonality defies the odds of nature

The ability to respond to natural selection under novel conditions is critical for the establishment and persistence of introduced alien species and their ability to become invasive. Here we correlated neutral and quantitative genetic diversity of the weed Pennisetum setaceum Forsk. Chiov. (Poaceae) with differing global (North American and African) patterns of invasiveness and compared this diversity to native range populations. Numerous molecular markers indicate complete monoclonality within and among all of these areas (F(ST) = 0.0) and is supported by extreme low quantitative trait variance (Q(ST) = 0.00065-0.00952). The results support the general-purpose-genotype hypothesis that can tolerate all environmental variation. However, a single global genotype and widespread invasiveness under numerous environmental conditions suggests a super-genotype. The super-genotype described here likely evolved high levels of plasticity in response to fluctuating environmental conditions during the Early to Mid Holocene. During the Late Holocene, when environmental conditions were predominantly constant but extremely inclement, strong selection resulted in only a few surviving genotypes.     

Micromorphology of fruits and seeds of Iranian Geranium (Geraniaceae), and its systematic significance

The fruit and seed micromorphology of 22 species of Geranium, representing the eight sections of the genus represented in Iran (G. sectt. Dissecta, Geranium, and Tuberosa of subgen. Geranium; sectt. Batrachioidea, Divaricata, Lucida, Ruberta and Trilopha of subgen. Robertium), have been examined by scanning electron microscopy (SEM). Macro‐ and micromorphological characters, including fruit and seed shape, size, color, hair type and density, mericarp ornamentations, hilum position, seed coat pattern, epidermal cell shape, and anticlinal and periclinal cell walls, are presented. Two microsculpturing patterns are recognized on the mericarp surface: reticulate and pusticulate. The micromorphology of the seed coat showed four distinctive cell patterns. The seed epidermis is constructed either of polygonal, elongated polygonal, or square to rectangular cells. The polygonal type is the most common among the studied species, but the variation in testa cell characters, their size and shape, may provide further information and useful diagnostic characters at specific and infraspecific rank. The shape and color of the seeds are, however, of little systematic value. Fruit characters were found to be important for separating taxa at infrageneric rank and our results show that the species can be separated into subgenera and sections based on fruit morphology.     

Varicella zoster virus ORF25 gene product: an essential hub protein linking encapsidation proteins and the nuclear egress complex

Varicella zoster virus (VZV) ORF25 is a 156 amino acid protein belonging to the approximately 40 core proteins that are conserved throughout the Herpesviridae. By analogy to its functional orthologue UL33 in Herpes simplex virus 1 (HSV-1), ORF25 is thought to be a component of the terminase complex. To investigate how cleavage and encapsidation of viral DNA links to the nuclear egress of mature capsids in VZV, we tested 10 VZV proteins that are predicted to be involved in either of the two processes for protein interactions against each other using three independent protein-protein interaction (PPI) detection systems: the yeast-two-hybrid (Y2H) system, a luminescence based MBP pull-down interaction screening assay (LuMPIS), and a bioluminescence resonance energy transfer (BRET) assay. A set of 20 interactions was consistently detected by at least 2 methods and resulted in a dense interaction network between proteins associated in encapsidation and nuclear egress. The results indicate that the terminase complex in VZV consists of ORF25, ORF30, and ORF45/42 and support a model in which both processes are closely linked to each other. Consistent with its role as a central hub for protein interactions, ORF25 is shown to be essential for VZV replication.     

A trade-off between embryonic development rate and immune function of avian offspring is revealed by considering embryonic temperature

Long embryonic periods are assumed to reflect slower intrinsic development that are thought to trade off to allow enhanced physiological systems, such as immune function. Yet, the relatively rare studies of this trade-off in avian offspring have not found the expected trade-off. Theory and tests have not taken into account the strong extrinsic effects of temperature on embryonic periods of birds. Here, we show that length of the embryonic period did not explain variation in two measures of immune function when temperature was ignored, based on studies of 34 Passerine species in tropical Venezuela (23 species) and north temperate Arizona (11 species). Variation in immune function was explained when embryonic periods were corrected for average embryonic temperature, in order to better estimate intrinsic rates of development. Immune function of offspring trades off with intrinsic rates of embryonic development once the extrinsic effects of embryonic temperatures are taken into account.     

The mouse primary visual cortex is a site of production and sensitivity to estrogens

The classic female estrogen, 17β-estradiol (E2), has been repeatedly shown to affect the perceptual processing of visual cues. Although gonadal E2 has often been thought to influence these processes, the possibility that central visual processing may be modulated by brain-generated hormone has not been explored. Here we show that estrogen-associated circuits are highly prevalent in the mouse primary visual cortex (V1). Specifically, we cloned aromatase, a marker for estrogen-producing neurons, and the classic estrogen receptors (ERs) ERα and ERβ, as markers for estrogen-responsive neurons, and conducted a detailed expression analysis via in-situ hybridization. We found that both monocular and binocular V1 are highly enriched in aromatase- and ER-positive neurons, indicating that V1 is a site of production and sensitivity to estrogens. Using double-fluorescence in-situ hybridization, we reveal the neurochemical identity of estrogen-producing and -sensitive cells in V1, and demonstrate that they constitute a heterogeneous neuronal population. We further show that visual experience engages a large population of aromatase-positive neurons and, to a lesser extent, ER-expressing neurons, suggesting that E2 levels may be locally regulated by visual input in V1. Interestingly, acute episodes of visual experience do not affect the density or distribution of estrogen-associated circuits. Finally, we show that adult mice dark-reared from birth also exhibit normal distribution of aromatase and ERs throughout V1, suggesting that the implementation and maintenance of estrogen-associated circuits is independent of visual experience. Our findings demonstrate that the adult V1 is a site of production and sensitivity to estrogens, and suggest that locally-produced E2 may shape visual cortical processing.     

Genetic diversity of the endangered endemic Microcycas calocoma (Miq.) A. DC (Zamiaceae, Cycadales): Implications for conservation

The diversity and genetic structure of seven populations of Microcycas calocoma (Miq.) A. DC, were analyzed by gel electrophoresis using 19 allozymes. The mean number of alleles per locus (A) was 1.49 and the percentage of polymorphic loci was relatively high (P = 48.09). The mean observed (H_o) and expected heterozygosity (H_e) were 0.20 and 0.17, respectively. The F-statistics revealed a high population structure (F_st = 0.34). Mean gene flow between population pairs was Nm = 0.96. Results were compared with those of other cycad species, and indicate that M. calocoma populations have become fragmented due to increasing pressures of habitat conversion and disturbance. Also, geographical isolation among populations has generated allele loss in relation to altitude. The establishment and maintenance of protected areas for in situ conservation is critical to preserve the high genetic diversity of M. calocoma. Conservation strategy guidelines have been specified.     

Waste-derived activated carbons for removal of ibuprofen from solution: Role of surface chemistry and pore structure

The removal of a widespread used drug (i.e., ibuprofen) from water was investigated using high valuable carbon adsorbents obtained from chemical and physical activation of a bioresource (cork) and a municipal waste (plastic). The waste-derived carbons outperformed the adsorption capacity of commercial carbonaceous adsorbents due to their adequate features for the removal of the targeted compound. Regarding the adsorption mechanism, the results obtained point out that ibuprofen retention is favored in activated carbons with basic surface properties. On the other hand, the textural features also play an important role; the presence of a transport pores network (i.e., mesopores) is crucial to ensure the accessibility to the inner porosity, and the microporosity must be large enough to accommodate the ibuprofen molecule. Specifically, adsorbents with a large fraction of ultramicropores (pore widths <0.7 nm) are not adequate to effectively remove ibuprofen.     

Identification of a new fatty acid synthesis-transport machinery at the peroxisomal membrane

The neurodegenerative disease X-linked adrenoleukodystrophy (X-ALD) is characterized by the abnormal accumulation of very long chain fatty acids. Mutations in the gene encoding the peroxisomal ATP-binding cassette half-transporter, adrenoleukodystrophy protein (ALDP), are the primary cause of X-ALD. To gain a better understanding of ALDP dysfunction, we searched for interaction partners of ALDP and identified binary interactions to proteins with functions in fatty acid synthesis (ACLY, FASN, and ACC) and activation (FATP4), constituting a thus far unknown fatty acid synthesis-transport machinery at the cytoplasmic side of the peroxisomal membrane. This machinery adds to the knowledge of the complex mechanisms of peroxisomal fatty acid metabolism at a molecular level and elucidates potential epigenetic mechanisms as regulatory processes in the pathogenesis and thus the clinical course of X-ALD.     

The interaction between human PEX3 and PEX19 characterized by fluorescence resonance energy transfer (FRET) analysis

The process of peroxisome biogenesis involves several PEX genes that encode the machinery required to assemble the organelle. Among the corresponding peroxins the interaction between PEX3 and PEX19 is essential for early peroxisome biogenesis. However, the intracellular site of this protein interaction is still unclear. To address this question by fluorescence resonance energy transfer (FRET) analysis, we engineered the enhanced yellow fluorescent protein (EYFP) to the C-terminus of PEX3 and the enhanced cyan fluorescent protein (ECFP) to the N-terminus of PEX19. Functionality of the fusion proteins was shown by transfection of human PEX3- and PEX19-deficient fibroblasts from Zellweger patients with tagged versions of PEX3 and PEX19. This led to reformation of import-competent peroxisomes in both cell lines previously lacking detectable peroxisomal membrane structures. The interaction of PEX3-EYFP with ECFP-PEX19 in a PEX3-deficient cell line during peroxisome biogenesis was visualized by FRET imaging. Although PEX19 was predominantly localized to the cytoplasma, the peroxisome was identified to be the main intracellular site of the PEX3-PEX19 interaction. Results were confirmed and quantified by donor fluorescence photobleaching experiments. PEX3 deletion proteins lacking the N-terminal peroxisomal targeting sequence (PEX3 34-373-EYFP) or the PEX19-binding domain located in the C-terminal half of the protein (PEX3 1-140-EYFP) did not show the characteristic peroxisomal localization of PEX3, but were mislocalized to the cytoplasm (PEX3 34-373-EYFP) or to the mitochondria (PEX3 1-140-EYFP) and did not interact with ECFP-PEX19. We suggest that FRET is a suitable tool to gain quantitative spatial information about the interaction of peroxins during the process of peroxisome biogenesis in single cells. These findings complement and extend data from conventional in vitro protein interaction assays and support the hypothesis of PEX3 being an anchor for PEX19 at the peroxisomal membrane.