Molecular and biochemical characterization of a novel intronic single point mutation in a Tunisian family with glycogen storage disease type III

Genetic deficiency of the glycogen debranching enzyme causes glycogen storage disease type III, an autosomal recessive inherited disorder. The gene encoding this enzyme is designated as AGL gene. The disease is characterized by fasting hypoglycemia, hepatomegaly, growth retardation, progressive myopathy and cardiomyopathy. In the present study, we present clinical features and molecular characterization of two consanguineous Tunisian siblings suffering from Glycogen storage disease type III. The full coding exons of the AGL gene and their corresponding exon–intron boundaries were amplified for the patients and their parents. Gene sequencing identified a novel single point mutation at the conserved polypyrimidine tract of intron 21 in a homozygous state (IVS21-8A>G). This variant cosegregated with the disease and was absent in 102 control chromosomes. In silico analysis using online resources showed a decreased score of the acceptor splice site of intron 21. RT-PCR analysis of the AGL splicing pattern revealed a 7 bp sequence insertion between exon 21 and exon 22 due to the creation of a new 3′ splice site. The predicted mutant enzyme was truncated by the loss of 637 carboxyl-terminal amino acids as a result of premature termination. This novel mutation is the first mutation identified in the region of Bizerte and the tenth AGL mutation identified in Tunisia. Screening for this mutation can improve the genetic counseling and prenatal diagnosis of GSD III.     

DNA‐based discrimination between the sibling species Aphis gossypii Glover and Aphis frangulae Kaltenbach

Abstract Morphologically similar species occur in various groups of insects, including aphid pests. In Europe, Aphis frangulae Kaltenbach and Aphis gossypii Glover (sometimes considered as subspecies) are differentiated usually on the basis of life cycle and host plant. We used a sexual population of A. frangulae collected on the primary host and samples of A. gossypii collected on cucurbits or cotton for the development of molecular markers. DNA sequence data for the gene encoding cytochrome b and for the barcode region of cytochrome oxidase I, as well as a length polymorphism for an intron in the sodium channel para‐type gene discriminated unambiguously between the two taxa. These markers were also used as identification keys for aphids collected on crops belonging to the Solanaceae. The cytochrome b marker differentiates host‐related Aphis gossypii haplotypes, and the para‐type gene intron might be suitable for the resolution of taxonomic problems in other aphid species complexes.     

Now I know my ABC. A systems neurochemistry and functional metabolomic approach to understanding the GABAergic system

Here, we describe use of a reductionist brain model, the brain tissue slice, to generate snapshots of functional metabolism in response to a pharmacological (GABAergic) perturbation. Tissue slices prepared from Guinea pig cerebral cortex were incubated for 1 h in the presence of [3-¹³C]-pyruvate and ligands with affinity for GABA receptors. The resultant patterns of ¹³C flux and metabolite levels were measured by ¹³C/¹H NMR spectroscopy, generating 'metabolic fingerprints' for each ligand. Effects of agonists and effectors at GABA receptors (A, B, and C types) were examined, compared to those of exogenous GABA and evaluated using multivariate statistical models. Data clusterings did not directly correlate with GABA receptor types but produced at least five distinct groups ranked according to their affinity for GABA. As our experimental model retains, to a large extent, the structure and function of normal brain tissue, the generated database can be used to assess GABAergic ligands and make unique inferences relevant to their modes of action in brain.     

Strong seasonal disequilibrium measured between the oxygen isotope signals of leaf and soil CO2 exchange

The oxygen isotope composition (δ¹⁸O) of atmospheric CO₂ is among a very limited number of tools available to constrain estimates of the biospheric gross CO₂ fluxes, photosynthesis and respiration at large scales. However, the accuracy of the partitioning strongly depends on the extent of isotopic disequilibrium between the signals carried by these two gross fluxes. Chamber‐based field measurements of total CO₂ and CO¹⁸O fluxes from foliage and soil can help evaluate and refine our models of isotopic fractionation by plants and soils and validate the extent and pattern of isotopic disequilibrium within terrestrial ecosystems. Owing to sampling limitations in the past, such measurements have been very rare and covered only a few days. In this study, we coupled automated branch and soil chambers with tuneable diode laser absorption spectroscopy techniques to continuously capture the δ¹⁸O signals of foliage and soil CO₂ exchange in a Pinus pinaster Aït forest in France. Over the growing season, we observed a seasonally persistent isotopic disequilibrium between the δ¹⁸O signatures of net CO₂ fluxes from leaves and soils, except during rain events when the isotopic imbalance became temporarily weaker. Variations in the δ¹⁸O of CO₂ exchanged between leaves, soil and the atmosphere were well explained by theory describing changes in the oxygen isotope composition of ecosystem water pools in response to changes in leaf transpiration and soil evaporation.     

Mites as biological tags of their hosts

Movements and spatial distribution of host populations are expected to shape the genetic structure of their parasite populations. Comparing the genetic patterns of both interacting species may improve our understanding of their evolutionary history. Moreover, genetic analyses of parasites with horizontal transmission may serve as indicators of historical events or current demographic processes that are not apparent in the genetic signature of their hosts. Here, we compared mitochondrial variation in populations of the ectoparasitic mite Spinturnix myoti with the genetic pattern of its host, the Maghrebian bat Myotis punicus in North Africa and in the islands of Corsica and Sardinia. Mite mitochondrial differentiation among populations was correlated with both host mitochondrial and nuclear differentiation, suggesting spatial co‐differentiation of the lineages of the two interacting species. Therefore our results suggest that parasite dispersal is exclusively mediated by host movements, with open water between landmasses as a main barrier for host and parasite dispersal. Surprisingly the unique presence of a continental European mite lineage in Corsica was inconsistent with host phylogeographical history and strongly suggests the former presence of European mouse‐eared bats on this island. Parasites may thus act as biological tags to reveal the presence of their now locally extinct host.     

Rapid Assessment of Avoidable Blindness in the Occupied Palestinian Territories

Background

There are no recent data on the prevalence and causes of blindness in the Occupied Palestinian Territories. The aim of our study was to estimate the prevalence and causes of blindness and visual impairment in the population aged 50 years and above in the Occupied Palestinian Territories using the Rapid Assessment of Avoidable Blindness (RAAB) survey method.

Methods and Findings

Clusters of 40 people who were 50 years and above were selected with probability proportionate to size using a multistage cluster random sampling method. Participants received a comprehensive ophthalmic examination in their homes, including visual acuity testing by one of three experienced ophthalmologists. The principal cause for visual loss was determined by an experienced ophthalmologist using portable diagnostic instruments. Information about previous cataract surgery, satisfaction with surgery and barriers to cataract surgery were collected. The prevalence of self-reported diabetes was also determined. The prevalence of bilateral blindness (VA<3/60 in the better eye with available correction) was 3.4% (95% CI: 2.7–4.0), 2.0% (95% CI: 1.4–2.5) for severe visual impairment (VA≥3/60 and <6/60), and 7.4% (95% CI: 6.4–8.3) for visual impairment (VA≥6/60 and <6/18). Avoidable causes (i.e. cataract, refractive error, aphakia, surgical complications, corneal scarring and phthysis) accounted for 80.0% of bilateral blindness, severe visual impairment (70.7%) and visual impairment (86.2%). Cataract was the main cause of blindness (55.0%). The prevalence of blindness was higher in Gaza (4.9%, 95% CI: 3.7–6.1%) than in the West Bank (2.5%, 95% CI: 1.9–3.1%) and among women (4.3%,95% CI: 3.3–5.2%) compared to men (2.2%,95%CI:1.5–2.9%). Among people who had undergone cataract surgery in the past, only 54.5% of eyes obtained a good outcome (VA≥6/18), 23.2% had a borderline outcome (VA<6/18 and ≥6/60) and 22.3% had a poor outcome (VA<6/60) with available correction. The prevalence of self-reported diabetes mellitus in ≥50 year age group was 26.4% (95% CI: 24.9–27.9).

Conclusions

The prevalence of blindness suggests that significant numbers of people in the Occupied Palestinian Territories exist who do not access eye care - predominantly women and those residing in Gaza. Programmes need to focus on maximizing the use of current services by these excluded groups.     

Human cytoplasmic serine hydroxymethyltransferase is an mRNA binding protein

The 5' untranslated region (UTR) of the human cytoplasmic serine hydroxymethyltransferase (cSHMT) message is alternatively spliced, creating a full-length 5' UTR (LUTR) encoded within exons 1-3 and a shorter UTR (SUTR) that results from excision of exon 2. The role of the 5' UTRs in cSHMT expression was investigated by fusing the cSHMT 5' UTRs to the 5' end of the luciferase gene. Human cSHMT protein at 10 microM inhibits in vitro translation of cSHMT 5' UTR-luciferase fusion mRNA templates by more than 90%, but does not inhibit translation of the luciferase message lacking the UTR. Translation inhibition is independent of amino acid and folate substrate binding to the cSHMT enzyme. The cSHMT SUTR-luciferase mRNA binds to the cSHMT.glycine.5-formyltetrahydrofolate ternary complex with an apparent K(d) of 10 microM. Gel mobility shift assays demonstrate that the human cSHMT protein binds to the cSHMT LUTR-luciferase fusion mRNA in the presence and absence of glycine and 5-formyltetrahydrofolate pentaglutamate. The fusion cSHMT SUTR-luciferase message at 65 microM inhibits the cSHMT-catalyzed cleavage of allothreonine as a partial mixed type inhibitor, reducing both k(cat) and K(m) by 40 and 75%, respectively, while tRNA has no effect on cSHMT catalysis. These studies indicate that the cSHMT protein can bind mRNA, and displays increased affinity for the 5' untranslated region of its mRNA.     

Arabidopsis species hybrids in the study of species differences and evolution of amphiploidy in plants

It is estimated that 5 million years of evolution separate Arabidopsis thaliana from its close relative Arabidopsis lyrata. The two taxa differ by many characteristics, and together they exemplify the differentiation of angiosperms into self-fertilizing and cross-fertilizing species as well as annual and perennial species. Despite their disparate life histories, the two species can be crossed to produce viable and vigorous hybrids exhibiting heterotic effects. Although pollen sterile, the hybrids produce viable ovules and were used as female parent in backcrosses to both parental species. The resulting backcross plants exhibited transgressive variation for a number of interesting developmental and growth traits as well as negative nuclear/cytoplasmic interactions. Moreover, the genesis of a fertile amphidiploid neospecies, apparently by spontaneous somatic doubling in an interspecific hybrid, was observed in the laboratory. The mechanisms responsible for the generation of amphiploids and the subsequent evolution of amphiploid genomes can now be studied through direct observation using the large arsenal of molecular tools available for Arabidopsis.