The Crown Pearl: a draft genome assembly of the European freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758)

Since historical times, the inherent human fascination with pearls turned the freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758) into a highly valuable cultural and economic resource. Although pearl harvesting in M. margaritifera is nowadays residual, other human threats have aggravated the species conservation status, especially in Europe. This mussel presents a myriad of rare biological features, e.g. high longevity coupled with low senescence and Doubly Uniparental Inheritance of mitochondrial DNA, for which the underlying molecular mechanisms are poorly known. Here, the first draft genome assembly of M. margaritifera was produced using a combination of Illumina Paired-end and Mate-pair approaches. The genome assembly was 2.4 Gb long, possessing 105,185 scaffolds and a scaffold N50 length of 288,726 bp. The ab initio gene prediction allowed the identification of 35,119 protein-coding genes. This genome represents an essential resource for studying this species’ unique biological and evolutionary features and ultimately will help to develop new tools to promote its conservation.     

Physicochemical Properties and Dissolution Studies of Dexamethasone Acetate-β-Cyclodextrin Inclusion Complexes Produced by Different Methods

Inclusion complexes between dexamethasone acetate (DMA), a poorly water soluble drug, and β-cyclodextrin (βCD) were obtained to improve the solubility and dissolution rate of this drug. Phase-solubility profile indicated that the solubility of DMA was significantly increased in the presence of βCD (33-fold) and was classified as A_L-type, indicating the 1:1 stoichiometric inclusion complexes. Solid complexes prepared by different methods (kneading, coevaporation, freeze drying) and physical mixture were characterized by differential scanning calorimetry, thermogravimetry, infrared absorption and optical microscopy. Preparation methods influenced the physicochemical properties of the products. The dissolution profiles of solid complexes were determined and compared with those DMA alone and their physical mixture, in three different mediums: simulated gastric fluid (pH 1.2), simulated intestinal fluid (pH 7.4) and distilled water. The dissolution studies showed that in all mediums DMA presented an incomplete dissolution even in four hours. In contrast, the complexes formed presented a higher dissolution rate in simulated gastric fluid (SGF pH 1.2), which indicate that these have different ionization characteristics. According to the results, the freeze–dried and kneaded products exhibited higher dissolution rates than the drug alone, in all the mediums.     

Genetic analysis of completely sequenced disease-associated MHC haplotypes identifies shuffling of segments in recent human history

The major histocompatibility complex (MHC) is recognised as one of the most important genetic regions in relation to common human disease. Advancement in identification of MHC genes that confer susceptibility to disease requires greater knowledge of sequence variation across the complex. Highly duplicated and polymorphic regions of the human genome such as the MHC are, however, somewhat refractory to some whole-genome analysis methods. To address this issue, we are employing a bacterial artificial chromosome (BAC) cloning strategy to sequence entire MHC haplotypes from consanguineous cell lines as part of the MHC Haplotype Project. Here we present 4.25 Mb of the human haplotype QBL (HLA-A26-B18-Cw5-DR3-DQ2) and compare it with the MHC reference haplotype and with a second haplotype, COX (HLA-A1-B8-Cw7-DR3-DQ2), that shares the same HLA-DRB1, -DQA1, and -DQB1 alleles. We have defined the complete gene, splice variant, and sequence variation contents of all three haplotypes, comprising over 259 annotated loci and over 20,000 single nucleotide polymorphisms (SNPs). Certain coding sequences vary significantly between different haplotypes, making them candidates for functional and disease-association studies. Analysis of the two DR3 haplotypes allowed delineation of the shared sequence between two HLA class II-related haplotypes differing in disease associations and the identification of at least one of the sites that mediated the original recombination event. The levels of variation across the MHC were similar to those seen for other HLA-disparate haplotypes, except for a 158-kb segment that contained the HLA-DRB1, -DQA1, and -DQB1 genes and showed very limited polymorphism compatible with identity-by-descent and relatively recent common ancestry (<3,400 generations). These results indicate that the differential disease associations of these two DR3 haplotypes are due to sequence variation outside this central 158-kb segment, and that shuffling of ancestral blocks via recombination is a potential mechanism whereby certain DR-DQ allelic combinations, which presumably have favoured immunological functions, can spread across haplotypes and populations.     

Precision mapping of the human O‐GalNAc glycoproteome through SimpleCell technology

Glycosylation is the most abundant and diverse posttranslational modification of proteins. While several types of glycosylation can be predicted by the protein sequence context, and substantial knowledge of these glycoproteomes is available, our knowledge of the GalNAc‐type O‐glycosylation is highly limited. This type of glycosylation is unique in being regulated by 20 polypeptide GalNAc‐transferases attaching the initiating GalNAc monosaccharides to Ser and Thr (and likely some Tyr) residues. We have developed a genetic engineering approach using human cell lines to simplify O‐glycosylation (SimpleCells) that enables proteome‐wide discovery of O‐glycan sites using ‘bottom‐up’ ETD‐based mass spectrometric analysis. We implemented this on 12 human cell lines from different organs, and present a first map of the human O‐glycoproteome with almost 3000 glycosites in over 600 O‐glycoproteins as well as an improved NetOGlyc4.0 model for prediction of O‐glycosylation. The finding of unique subsets of O‐glycoproteins in each cell line provides evidence that the O‐glycoproteome is differentially regulated and dynamic. The greatly expanded view of the O‐glycoproteome should facilitate the exploration of how site‐specific O‐glycosylation regulates protein function.     

Copper(II) complexes with (2-hydroxybenzyl-2-pyridylmethyl)amine–Hbpa: syntheses, characterization and crystal structures of the ligand and [Cu(II)(Hbpa)2](ClO4)2·2H2O

Copper(II) complexes were synthesized and characterized by means of elemental analysis, IR and visible spectroscopies, EPR and electrochemistry, as well as X-ray structure crystallography. The group consists of discrete mononuclear units with the general formula [Cu(II)(Hbpa)₂](A)₂·nH₂O, where Hbpa=(2-hydroxybenzyl-2-pyridylmethyl)amine and A=ClO₄ ⁻, n=2 (1), CH₃COO⁻, n=3 (2), NO₃ ⁻, n=2 (3) and SO₄ ²⁻, n=3 (4). The structures of the ligand Hbpa and complex 1 have been determined by X-ray crystallography. Complexes 1–4 have had their UV–Vis spectra measured in both MeCN and DMF. It was observed that the compounds interact with basic solvents, such that molecules coordinate to the metal in axial positions in which phenol oxygen atoms are coordinated in the protonated forms. The values were all less than 1000 M⁻¹ cm⁻¹. EPR measurements on powdered samples of 1–3 gave g/A values between 105 and 135 cm⁻¹, typical for square planar coordination environments. Complex 4·3H₂O exhibits a behaviour typical for tetrahedral coordination. The electrochemical behaviour for complexes 1 and 2 was studied showing irreversible redox waves for both compounds.     

Comparative proteomic analysis of growth hormone secretagogue A233 treatment of murine macrophage cells J774A.2 indicates it has a role in antiviral innate response

BackgroundGrowth hormone secretagogues (GHS), among other factors, regulate the release of GH. The biological activity of the secretagogue peptide A233 as a promoter of growth and innate immunity in teleost fish has previously been demonstrated, but its role in the immune system of mammals is not well understood.MethodsThe effect of the peptide was investigated in J774A.2 macrophage cells using a comparative proteomics approach after 6 and 12 h of peptide stimulation.ResultsThe functional analysis of differentially modulated proteins showed that A233 peptide treatment appears to promote activation and ROS-dependent cytotoxic functions in macrophages and enhanced expression of antiviral protein complexes such as MAVS. In accordance with this hypothesis, we found that A233 treatment enhanced superoxide anion production and the IFN-γ level in J774A.2 cells and mouse splenocytes, respectively, and reduced viral load in a dengue virus mouse model of infection.ConclusionsThe growth hormone secretagogue A233 peptide promotes activation of ROS-dependent cytotoxic functions and exerts immunomodulatory effects that enable an antiviral state in a dengue virus mouse model.General SignificanceThe increase of IFN-γ level and the differential modulation of antiviral proteins by the A233 peptide suggest that the molecule could activate an innate immune response with a possible further impact in the treatment of acute and chronic diseases.     

mGluR long‐term depression regulates GluA2 association with COPII vesicles and exit from the endoplasmic reticulum

mGluR long‐term depression (mGluR‐LTD) is a form of synaptic plasticity induced at excitatory synapses by metabotropic glutamate receptors (mGluRs). mGluR‐LTD reduces synaptic strength and is relevant to learning and memory, autism, and sensitization to cocaine; however, the mechanism is not known. Here we show that activation of Group I mGluRs in medium spiny neurons induces trafficking of GluA2 from the endoplasmic reticulum (ER) to the synapse by enhancing GluA2 binding to essential COPII vesicle proteins, Sec23 and Sec13. GluA2 exit from the ER further depends on IP3 and Ryanodine receptor‐controlled Ca²⁺ release as well as active translation. Synaptic insertion of GluA2 is coupled to removal of high‐conducting Ca²⁺‐permeable AMPA receptors from synapses, resulting in synaptic depression. This work demonstrates a novel mechanism in which mGluR signals release AMPA receptors rapidly from the ER and couple ER release to GluA2 synaptic insertion and GluA1 removal.     

Purification of betulinic acid from Eugenia florida (Myrtaceae) by high-speed counter-current chromatography

A high yield of betulinic acid (up to 17% from the ethanolic extract) was found in the leaves of Eugenia florida collected in south-eastern Brazil, making this species a potential commercial source of the title compound. Extracts of E. florida were subjected to solvent partition, and rapid high-speed counter-current chromatography (HSCCC) was applied to the semi-crude extracts to afford betulinic acid in high purity. The mobile and stationary phases were derived from the two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (10:5:2.5:1). The developing solvent system (stationary and mobile phases) for optimum HSCCC separation was chosen by dissolving the fraction to be chromatographed in the proposed solvent mixture and determining the amount of betulinic acid in each phase by densitometric TLC. Purified betulinic acid was characterized by 13C-NMR, GC-MS and co-injection of its methyl ester with standards in GC-FID. The HSCCC technique is commonly employed to isolate triterpene glycosides, but is applied in this study to an aglycone.     

Effect of acute hypoxia on microcirculatory and tissue oxygen levels in rat cremaster muscle.

Repeated exposure to brief periods of hypoxia leads to pathophysiological changes in experimental animals similar to those seen in sleep apnea. To determine the effects of such exposure on oxygen levels in vivo, we used an optical method to measure PO2 in microcirculatory vessels and tissue of the rat cremaster muscle during a 1-min step reduction of inspired oxygen fraction from 0.21 to 0.07. Under control conditions, PO2 was 98.1 +/- 1.9 Torr in arterial blood, 52.2 +/- 2.8 Torr in 29.0 +/- 2.7-microm arterioles, 26.8 +/- 1.7 Torr in the tissue interstitium near venous capillaries, and 35.1 +/- 2.6 Torr in 29.7 +/- 1.9-microm venules. The initial fall in PO2 during hypoxia was significantly greater in arterial blood, being 93% complete in the first 10 s, whereas it was 68% complete in arterioles, 47% at the tissue sites, and 38% in venules. In the 10- to 30-s period, the fall in normalized tissue and venular PO2 was significantly greater than in arterial PO2. At the end of hypoxic exposure, PO2 at all measurement sites had fallen very nearly in proportion to that in the inspired gas, but tissue oxygen levels did not reach critical PO2. Significant differences in oxyhemoglobin desaturation rate were also observed between arterial and microcirculatory vessels during hypoxia. In conclusion, the fall in microcirculatory and tissue oxygen levels in resting skeletal muscle is significantly slower than in arterial blood during a step reduction to an inspired oxygen fraction of 0.07, and tissue PO2 does not reach anaerobic levels.     

Evolutionary and structural analysis of the cytochrome c oxidase subunit I (COI) gene from Haematobia irritans, Stomoxys calcitrans and Musca domestica (Diptera: Muscidae) mitochondrial DNA.

This work describes the molecular characterization of the cytochrome c oxidase subunit I (COI) gene of the mitochondrial DNA from three species of great medical and veterinary importance: the horn fly, Haematobia irritans, the stable fly, Stomoxys calcitrans and the house fly, Musca domestica (Diptera: Muscidae) (Linnaeus). The nucleotide sequence in all species was 1536 bp in size and coded for a 512 amino acid peptide. The nucleotide bias for an A+T-rich sequence is linked to three features: a high A+T content throughout the entire gene, a high A+T content in the third codon position, and a predominance of A+T-rich codons. An anomalous TCG (serine) start codon was identified. Comparative analysis among members of the Muscidae, Scatophagidae, Calliphoridae and Drosophilidae showed high levels of nucleotide sequence conservation. Analysis of the divergent amino acids and COI protein topologies among these three Muscidae species agreed with the evolutionary model suggested for the insect mitochondrial COI protein. The characterization of the structure and evolution of this gene could be informative for further evolutionary analysis of dipteran species.