Autosomal-Recessive Intellectual Disability with Cerebellar Atrophy Syndrome Caused by Mutation of the Manganese and Zinc Transporter Gene SLC39A8

Manganese (Mn) and zinc (Zn) are essential divalent cations used by cells as protein cofactors; various human studies and animal models have demonstrated the importance of Mn and Zn for development. Here we describe an autosomal-recessive disorder in six individuals from the Hutterite community and in an unrelated Egyptian sibpair; the disorder is characterized by intellectual disability, developmental delay, hypotonia, strabismus, cerebellar atrophy, and variable short stature. Exome sequencing in one affected Hutterite individual and the Egyptian family identified the same homozygous variant, c.112G>C (p.Gly38Arg), affecting a conserved residue of SLC39A8. The affected Hutterite and Egyptian individuals did not share an extended common haplotype, suggesting that the mutation arose independently. SLC39A8 is a member of the solute carrier gene family known to import Mn, Zn, and other divalent cations across the plasma membrane. Evaluation of these two metal ions in the affected individuals revealed variably low levels of Mn and Zn in blood and elevated levels in urine, indicating renal wasting. Our findings identify a human Mn and Zn transporter deficiency syndrome linked to SLC39A8, providing insight into the roles of Mn and Zn homeostasis in human health and development.     

Fire persistence traits can be used to predict vegetation response to changing fire regimes at expansive landscape scales – an Australian example

Aim Building on a substantial literature addressing the fire responses of woody plants, particularly under mediterranean climates, we assess the extent to which fire persistence traits can be used to predict vegetation responses to fire regime changes in fire‐prone arid and savanna landscape settings. Location Australia, applying data from arid central to monsoonal northern regions (11–26° S, 129–138° E). Methods With reference to a substantial sub‐continental floristics dataset, we first assigned the fire response (obligate seeder, resprouter) and seedbank persistence (transient, dormant) of rapid and longer‐maturing (> 3 years) woody taxa. Using logistic regression, we then modelled the proportions of taxa possessing these traits as a function of mean annual rainfall (highly correlated with fire frequency) and terrain roughness (a measure of topographic variability) in 0.25° × 0.25° and 1° × 1° grid cells. Separate assessments were undertaken with datasets for 1264 sclerophyll and 236 rain forest taxa. Results This woody flora is characterized by taxa exhibiting mostly resprouting and dormant seedbank traits that promote site persistence. While numbers of obligate seeder and resprouter taxa were related positively to both rainfall and roughness, the relative abundance of both sclerophyll and rain forest obligate seeders decreased significantly with rainfall. The relative abundance of sclerophyll (especially long‐lived) obligate seeders alone increased with topographic roughness. The proportion of taxa with transient seedbanks increased with rainfall in resprouters generally, and in rain forest obligate seeders alone. Main conclusions We find that resprouters are favoured on more productive, fire‐prone sites, and obligate seeders are favoured in less productive, more fire‐protected settings. Seedbank persistence responses are more variable. These findings concur generally with theoretical constructs, and support comparable assessments in Australian and other fire‐prone systems ranging from mediterranean to boreal environments. Our observations illustrate that resprouting and obligate seeding syndromes, but not necessarily seedbank persistence, are useful predictors of vegetation responses to changing fire regime conditions at large landscape scales.     

Mapping the cofilin binding site on yeast G-actin by chemical cross-linking

Cofilin is a major cytoskeletal protein that binds to both monomeric actin (G-actin) and polymeric actin (F-actin) and is involved in microfilament dynamics. Although an atomic structure of the G-actin-cofilin complex does not exist, models of the complex have been built using molecular dynamics simulations, structural homology considerations, and synchrotron radiolytic footprinting data. The hydrophobic cleft between actin subdomains 1 and 3 and, alternatively, the cleft between actin subdomains 1 and 2 have been proposed as possible high-affinity cofilin binding sites. In this study, the proposed binding of cofilin to the subdomain 1/subdomain 3 region on G-actin has been probed using site-directed mutagenesis, fluorescence labeling, and chemical cross-linking, with yeast actin mutants containing single reactive cysteines in the actin hydrophobic cleft and with cofilin mutants carrying reactive cysteines in the regions predicted to bind to G-actin. Mass spectrometry analysis of the cross-linked complex revealed that cysteine 345 in subdomain 1 of mutant G-actin was cross-linked to native cysteine 62 on cofilin. A cofilin mutant that carried a cysteine substitution in the α3-helix (residue 95) formed a cross-link with residue 144 in actin subdomain 3. Distance constraints imposed by these cross-links provide experimental evidence for cofilin binding between actin subdomains 1 and 3 and fit a corresponding docking-based structure of the complex. The cross-linking of the N-terminal region of recombinant yeast cofilin to actin residues 346 and 374 with dithio-bis-maleimidoethane (12.4 Å) and via disulfide bond formation was also documented. This set of cross-linking data confirms the important role of the N-terminal segment of cofilin in interactions with G-actin.     

Flow cytometric detection of proteolysis in peptide libraries synthesised on optically encoded supports

The concept of optically encoding particles for solid phase organic synthesis has existed in the literature for several years. However, there remains a significant challenge to producing particles that are capable of withstanding harsh solvents and reagents whilst maintaining the integrity and range of the optical encoding. In this study, a new generation of fluorescently encoded support particles was used for both solid phase peptide synthesis and on-particle analysis of proteolysis in a multiplexed, flow cytometric assay. The success of the assay was demonstrated through the use of a model protease, trypsin. Our results show that the use of solid supports with high peptide yield, high swellability in water and high penetration of the enzyme into the interior of the particle is not absolutely necessary for proteolysis assays.     

The effect of vapocoolant spray on pain due to intravenous cannulation in children: a randomized controlled trial

Background

Established noninvasive pharmacologic means of alleviating pain and anxiety in children undergoing intravenous cannulation are time-consuming, and thus impractical for routine use in the emergency department. Vapocoolant sprays provide transient skin anesthesia within seconds of application. We compared the effect of a new vapocoolant spray to placebo on pain due to intravenous cannulation in children.

Methods

In this double-blind randomized controlled trial, which we conducted between June 1 and Sept. 12, 2006, 80 children aged 6–12 years received either vapocoolant spray or placebo before cannulation. Children rated their pain using a 100-mm colour visual analogue scale. Secondary outcomes included success rate on first attempt at cannulation and pain ratings by the children''s parents, nurses and child life specialists.

Results

We found a modest but significant reduction in pain with the use of vapocoolant spray (mean difference 19 mm, 95% confidence interval [CI] 6–32 mm; p < 0.01). Cannulation on first attempt was more often successful with the use of vapocoolant spray (85.0%) than with placebo (62.5%) (mean difference 22.5%, 95% CI 3.2%–39.9%; p = 0.03). The number needed to treat to prevent 1 cannulation failure was 5 (95% CI 3–32). Parents (p = 0.04), nurses (p = 0.01) and child life specialists (p < 0.01) considered the children''s pain to be reduced with the use of vapocoolant spray.

Interpretation

The vapocoolant spray in our study quickly and effectively reduced pain due to intravenous cannulation in children and improved the success rate of cannulation. It is an important option to reduce childhood procedural pain in emergency situations, especially when time precludes traditional interventions.(http://ClinicalTrials.gov trial register no. {"type":"clinical-trial","attrs":{"text":"NCT00130650","term_id":"NCT00130650"}}NCT00130650.)Children frequently undergo intravenous cannulation in the emergency department. This painful procedure causes considerable stress and anxiety for children and their parents.¹ Failure to alleviate pain results in an uncooperative child, unsuccessful procedures (increased reattempts), prolonged procedure time and dissatisfaction with care for all involved.² Although pain may be reduced by behavioural and pharmacologic interventions (e.g., age-appropriate patient preparation, parental presence, distraction techniques, subcutaneous local anesthetics, topical anesthetic and systemic anesthetics), most of these preparations are impractical in nonelective settings because they are too time-consuming.^3–12 Novel delivery approaches and new medication compositions may decrease this time.^13,14 Vapocoolant sprays are rapid-acting alternatives to topical anesthetics. They provide transient anesthesia via evaporation-induced skin cooling, which reduces pain. Results from studies of earlier vapocoolant sprays indicated that they reduced pain due to vaccine injection in children and adults,^15–18 but not pain due to intravenous cannulation in children.^19,20 We sought to determine whether a new product, Pain Ease (Gebauer Company, Cleveland, Ohio), would reduce pain with intravenous cannulation in children.     

A new endogenous form of PYY isolated from canine ileum: Gly-extended PYY(1-36)

We purified and identified the peptide YY (PYY) forms present and determined their levels from a portion of the canine ileum directly adjacent to the cecum by a new extraction method designed to prevent and evaluate degradation of endogenous peptides. We used three reverse phase chromatography steps with radioimmunoassay of fractions for PYY-like-immunoreactivity (PYY-LI). The purified fractions underwent intact protein/peptide mass spectrometry identification and sequencing (i.e. "top-down" MS analysis). This analysis confirmed the identity of a new form of PYY, PYY(1-36)-Gly, which co-elutes with PYY(1-36)-NH(2) through all three of separation steps used. The PYY(1-36)-Gly form represents approximately 20% of the total PYY found in this region of the canine intestine. In addition, we also found that the PYY(3-36)-NH(2) form represents 6% of the total PYY in the canine ileo-cecal junction. The physiological implication of the Gly-extended form of PYY(1-36) warrants further investigation.     

Technologically indicative properties of straw fractions of flax, linseed (Linum usitatissimum L.) and fibre hemp (Cannabis sativa L.)

In this study of the behaviour of the fractions of unretted and frost-retted fibre straws in damp air, a production scale method to separate fibre and shive from fibre plants was introduced and tested on bast fibre plants (Linum usitatissimum L. and Cannabis sativa L.). The method consists of optional drying of stalks, unloading bales, milling the straws with a hammer mill, separating the fractions from air stream with a cyclone and finally separating fibres from shives with a screening drum. Fractions were characterized focusing on technologically indicative properties such as equilibrium moisture content, ash and microbiological quality. Unretted fractions of the bast fibre plant stem reached higher equilibrium moisture contents than the retted fractions, and hemp fibres absorbed more moisture from air than did the Linum fibres. In very humid air, all fractions began to lose weight due to moulding. The weight decrease during the first week was lower in frost-retted than in unretted fractions. The frost-retted fractions appeared to be more resistant to humidity in the short term. The total number of microbes and especially the numbers of yeasts and moulds can be used as a criterion of hygienic level. For green fractions, the mould level was similar in fibres and in shives, but frost-retted shives contained more moulds than the unretted shives. The mould content of a fraction had no direct correlation with the moulding tendency of the fraction. The ash contents of fibres were somewhat higher than those of shives, due to a probable soil contamination. Ash content did not have significant correlation with microbiological quality, although ash is a possible risk factor for hygienic quality. According to the results of this study it is highly important to study the quality of the production chain of bast fibre plants to ensure the quality of industrial products. From the producer's point of view, raw material with defined quality can be directed to the most suitable application. The behaviour of fractions in various ambient atmospheres, and other quality aspects such as hygienic level can be used as criteria for defining the most appropriate product applications.     

N-Acetyl Aspartic Acid (NAA) and N-Acetyl Aspartylglutamic Acid (NAAG) in Human Ventricular,Subarachnoid, and Lumbar Cerebrospinal Fluid

N-Acetylaspartic and N-acetylaspartylglutamic acid concentrations in human ventricular, subarachnoid and lumbar cerebrospinal fluid were measured by combined gas chromatography-mass spectrometry using selected ion monitoring with deuterated internal standards. N-Acetylaspartate concentrations were in the range 55, 9, and 1 M, respectively; N-acetylaspartylglutamate concentrations in the same fluids were in the range 8, 3 and 4 M, respectively. There did not appear to be any difference in lumbar fluid concentrations of either compound between control subjects, schizophrenic patients, Alzheimer's disease patients and a pooled group of patients with neurological degeneration. Ventricular concentrations of both compounds were greatly increased in deceased patients suggesting that maintenance of their intracellular concentrations is probably energy dependent. The concentrations of these compounds in lumbar cerebrospinal fluid from living, and ventricular cerebrospinal fluid from deceased subjects were weakly correlated with one another. In lumbar fluid neither compound appeared to be correlated with age. Analysis of serially collected lumbar samples from two subjects showed a weak concentration gradient for both compounds. Neither antipsychotic medication nor the acid transport inhibitor probenecid had any effect on lumbar concentrations of either compound. Attempts to use anion exchange high pressure liquid chromatography with UV detection for measurement of the low concentrations of N-acetylaspartate found in cerebrospinal fluid from living subjects were unsuccessful.