The activation of the decapping enzyme DCP2 by DCP1 occurs on the EDC4 scaffold and involves a conserved loop in DCP1

The removal of the 5′-cap structure by the decapping enzyme DCP2 and its coactivator DCP1 shuts down translation and exposes the mRNA to 5′-to-3′ exonucleolytic degradation by XRN1. Although yeast DCP1 and DCP2 directly interact, an additional factor, EDC4, promotes DCP1–DCP2 association in metazoan. Here, we elucidate how the human proteins interact to assemble an active decapping complex and how decapped mRNAs are handed over to XRN1. We show that EDC4 serves as a scaffold for complex assembly, providing binding sites for DCP1, DCP2 and XRN1. DCP2 and XRN1 bind simultaneously to the EDC4 C-terminal domain through short linear motifs (SLiMs). Additionally, DCP1 and DCP2 form direct but weak interactions that are facilitated by EDC4. Mutational and functional studies indicate that the docking of DCP1 and DCP2 on the EDC4 scaffold is a critical step for mRNA decapping in vivo. They also revealed a crucial role for a conserved asparagine–arginine containing loop (the NR-loop) in the DCP1 EVH1 domain in DCP2 activation. Our data indicate that DCP2 activation by DCP1 occurs preferentially on the EDC4 scaffold, which may serve to couple DCP2 activation by DCP1 with 5′-to-3′ mRNA degradation by XRN1 in human cells.     

Large DNA inversions,deletions, and TaqI site mutations in severe haemophilia A

Large DNA inversions caused by an intrachromosomal recombination between homologous regions located in intron 22 and 5 of the factor VIII gene have recently been identified in patients with severe haemophilia A. To evaluate better the prevalence of this large inversion and to estimate the overall sensitivity of the Southern blot/hybridization method we analysed the factor VIII gene of 49 unrelated patients with severe haemophilia A. All patients were screened for the inversion mutation, TaqI site mutations, and deletions. Mutations were identified in 31 (63%) patients, and comprised 24 large inversions, 4 partial deletions, and 3 point mutations. Three different haplotypes were characterised in the patients presenting the inversion mutation, confirming its independent origin. Two novel deletions are reported: a large one spanning from intron 14 to intron 22 and a deletion of 86 bp comprising the 3 region of exon 1 and 39–41 bp of intron 1. DNA sequencing of the deletion junction showed no significant homology between normal 5 and 3 sequences around the breakpoints. A novel missense mutation is also reported: CGAGGA, Arg-2209 to Gly. These results confirm that the inversion mutation is the most common cause of severe haemophilia A and indicate that the Southern blot/hybridization assay should be used as the first method for screening of mutations in severe haemophilia A.     

Loss of functionally important and regionally endemic species from streams forced into intermittency by global warming

Climate change is altering hydrological cycles globally, and in Mediterranean (med-) climate regions it is causing the drying of river flow regimes, including the loss of perennial flows. Water regime exerts a strong influence over stream assemblages, which have developed over geological timeframes with the extant flow regime. Consequently, sudden drying in formerly perennial streams is expected to have large, negative impacts on stream fauna. We compared contemporary (2016/17) macroinvertebrate assemblages of formerly perennial streams that became intermittently flowing (since the early 2000s) to assemblages recorded in the same streams by a study conducted pre-drying (1981/82) in the med-climate region of southwestern Australia (the Wungong Brook catchment, SWA), using a multiple before-after, control-impact design. Assemblage composition in the stream reaches that remained perennial changed very little between the studies. In contrast, recent intermittency had a profound effect on species composition in streams impacted by drying, including the extirpation of nearly all Gondwanan relictual insect species. New species arriving at intermittent streams tended to be widespread, resilient species including desert-adapted taxa. Intermittent streams also had distinct species assemblages, due in part to differences in their hydroperiods, allowing the establishment of distinct winter and summer assemblages in streams with longer-lived pools. The remaining perennial stream is the only refuge for ancient Gondwanan relict species and the only place in the Wungong Brook catchment where many of these species still persist. The fauna of SWA upland streams is becoming homogenised with that of the wider Western Australian landscape, as drought-tolerant, widespread species replace local endemics. Flow regime drying caused large, in situ alterations to stream assemblage composition and demonstrates the threat posed to relictual stream faunas in regions where climates are drying.     

Heritability of adult body height: a comparative study of twin cohorts in eight countries.

A major component of variation in body height is due to genetic differences, but environmental factors have a substantial contributory effect. In this study we aimed to analyse whether the genetic architecture of body height varies between affluent western societies. We analysed twin data from eight countries comprising 30,111 complete twin pairs by using the univariate genetic model of the Mx statistical package. Body height and zygosity were self-reported in seven populations and measured directly in one population. We found that there was substantial variation in mean body height between countries; body height was least in Italy (177 cm in men and 163 cm in women) and greatest in the Netherlands (184 cm and 171 cm, respectively). In men there was no corresponding variation in heritability of body height, heritability estimates ranging from 0.87 to 0.93 in populations under an additive genes/unique environment (AE) model. Among women the heritability estimates were generally lower than among men with greater variation between countries, ranging from 0.68 to 0.84 when an additive genes/shared environment/unique environment (ACE) model was used. In four populations where an AE model fit equally well or better, heritability ranged from 0.89 to 0.93. This difference between the sexes was mainly due to the effect of the shared environmental component of variance, which appears to be more important among women than among men in our study populations. Our results indicate that, in general, there are only minor differences in the genetic architecture of height between affluent Caucasian populations, especially among men.     

High-performance liquid chromatographic method for the determination of mycophenolate mofetil in human plasma

A method for the quantification of mycophenolate mofetil (MMF, CellCept) in plasma using solid-phase extraction and HPLC is described here. A solution of internal standard is added to a 0.5-ml plasma aliquot. The resulting sample is treated with water and dilute HCl and applied to a C₁₈ solid-phase extraction column. After a water wash, the MMF and internal standard are eluted with methanol-0.1 M citrate-phosphate buffer, pH 2.6 (80:20, v/v). A 20-μl aliquot of the eluate is injected onto a C₁₈ column (5 μm particle size, 150 × 4.6 mm I.D.) and eluted at ambient temperature with acetonitrile-0.05 M citrate-phosphate buffer, pH 3.6, containing 0.02 M heptanesulfonic acid (41:59, v/v). Quantification is achieved by UV detection at 254 nm. The method is reproducible, accurate and specific for MMF. Using 0.5 ml of plasma for analysis, the quantification limit is 0.400 μg/ml and the range is 0.400–20 μg/ml. Based on the stability profile of MMF in plasma, it is recommended that blood samples collected following intravenous infusion be immediately stored on ice and that plasma be prepared rapidly, immediately stored frozen at −80°C and analyzed within four months of collection.     

The elongation of amylose and amylopectin chains in isolated starch granules

The aim of this work was to investigate the conditions required for amylose synthesis in starch granules. Although the major granule-bound isoform of starch synthase - GBSSI - catalyses the synthesis of amylose in vivo, ¹⁴C from ADP[¹⁴C]glucose was incorporated primarily into a specific subset of amylopectin chains when supplied to starch granules isolated from pea (Pisum sativum L.) embryos and potato (Solanum tuberosum L.) tubers. Incubation of granules with soluble extracts of these organs revealed that the extracts contained compounds that increased the incorporation of ¹⁴C into amylose. These compounds were rendered inactive by treatment of the extracts with α-glucosidase, suggesting that they were malto-oligosaccharides. Consistent with this idea, provision of pure malto-oligosaccharides to isolated granules resulted in a dramatic shift in the pattern of incorporation of ¹⁴C, from amylopectin chains to amylose molecules. Comparison of the pattern of incorporation in granules from wild-type peas and lam mutant peas which lack GBSSI showed that this effect of malto-oligosaccharides was specifically on GBSSI. The significance of these results for understanding of the synthesis of amylose and amylopectin in storage organs is discussed.     

Control of trophoblast cell differentiation: Lessons from the genetics of early pregnancy loss and trophoblast neoplasia

Trophoblast cell differentiation is crucial to the morphogenesis of the placenta and thus the establishment of pregnancy and the growth and development of the embryo/fetus. In the present review, we discuss current evidence for the existence of regulatory genes crucial to trophoblast cell differentiation and placental morphogenesis. The elucidation of regulatory pathways controlling normal differentiation of trophoblast cells will facilitate the identification of sensitive junctures in the regulatory pathways leading to various developmental disorders, including those associated with the initiation of pregnancy, fetal growth retardation and gestational trophoblast disease.     

Lack of genetic diversity across diverse immune genes in an endangered mammal,the Tasmanian devil (Sarcophilus harrisii)

The Tasmanian devil (Sarcophilus harrisii) is threatened with extinction due to the spread of devil facial tumour disease. Polymorphisms in immune genes can provide adaptive potential to resist diseases. Previous studies in diversity at immune loci in wild species have almost exclusively focused on genes of the major histocompatibility complex (MHC); however, these genes only account for a fraction of immune gene diversity. Devils lack diversity at functionally important immunity loci, including MHC and Toll‐like receptor genes. Whether there are polymorphisms at devil immune genes outside these two families is unknown. Here, we identify polymorphisms in a wide range of key immune genes, and develop assays to type single nucleotide polymorphisms (SNPs) within a subset of these genes. A total of 167 immune genes were examined, including cytokines, chemokines and natural killer cell receptors. Using genome‐level data from ten devils, SNPs within coding regions, introns and 10 kb flanking genes of interest were identified. We found low polymorphism across 167 immune genes examined bioinformatically using whole‐genome data. From this data, we developed long amplicon assays to target nine genes. These amplicons were sequenced in 29–220 devils and found to contain 78 SNPs, including eight SNPS within exons. Despite the extreme paucity of genetic diversity within these genes, signatures of balancing selection were exhibited by one chemokine gene, suggesting that remaining diversity may hold adaptive potential. The low functional diversity may leave devils highly vulnerable to infectious disease, and therefore, monitoring and preserving remaining diversity will be critical for the long‐term management of this species. Examining genetic variation in diverse immune genes should be a priority for threatened wildlife species. This study can act as a model for broad‐scale immunogenetic diversity analysis in threatened species.     

A porous medium‐chain‐length poly(3‐hydroxyalkanoates)/hydroxyapatite composite as scaffold for bone tissue engineering

Polyhydroxyalkanoates (PHA) are hydrophobic biopolymers with huge potential for biomedical applications due to their biocompatibility, excellent mechanical properties and biodegradability. A porous composite scaffold made of medium‐chain‐length poly(3‐hydroxyalkanoates) (mcl‐PHA) and hydroxyapatite (HA) was fabricated using particulate leaching technique and NaCl as a porogen. Different percentages of HA loading was investigated that would support the growth of osteoblast cells. Ultrasonic irradiation was applied to facilitate the dispersion of HA particles into the mcl‐PHA matrix. The different P(3HO‐co‐3HHX)/HA composites were investigated using field emission scanning electron microscopy (FESEM), X‐ray diffraction (XRD) and energy dispersive X‐ray analysis (EDXA). The scaffolds were found to be highly porous with interconnecting pore structures and the HA particles were homogeneously dispersed in the polymer matrix. The scaffolds biocompatibility and osteoconductivity were also assessed following the proliferation and differentiation of osteoblast cells on the scaffolds. From the results, it is clear that scaffolds made from P(3HO‐co‐3HHX)/HA composites are viable candidate materials for bone tissue engineering applications.