Paroxysmal and cognitive phenotypes in Prrt2 mutant mice

Mutations in proline‐rich transmembrane protein 2 (PRRT2) cause a range of episodic disorders that include paroxysmal kinesigenic dyskinesia and benign familial infantile epilepsy. Mutations are generally loss of function and include the c649dupC frameshifting mutation that is present in around 80% of affected individuals. To investigate how Prrt2 loss of function mutations causes disease, we performed a phenotypic investigation of a transgenic Prrt2 knockout (Prrt2 KO) mouse. We observed spontaneous paroxysmal episodes with behavioural features of both seizure and movement disorders, as well as unexplained deaths in KO and HET animals. KO mice showed spatial learning deficits in the Morris water maze, as well as gait abnormalities in the quantitative Digigait analysis; both of which may be representative of the more severe phenotypes experienced by homozygous patients. These findings extend the described phenotypes of Prrt2 mutant mice, further confirming their utility for in vivo investigation of the role of Prrt2 mutations in episodic diseases.     

The influence of organic and inorganic chelators on the toxicity of bulk and nanoparticles of zinc oxide during germination and seedling growth of Nicotiana tabacum L.

Nano and bulk-forms of zinc oxide (ZnO) are used extensively in industry and consequently may accumulate in the environment. However, there is little information available on the comparative effects of these forms during the critical early stages of plant life. Furthermore, the role of chelating agents, which affect the bioavailability of metals, in ameliorating plant stress due to exposure to nano and bulk-forms of ZnO is not well characterised. In this study, the effects of different concentrations (0.5, 2.5, 5, 10, 50 and 100 ppm) of nano ZnO (22 nm) and bulk ZnO (natural form, 1000 nm) with and without organic (citrate) and inorganic (EDTA) chelators on germination and seedling growth, and oxidative stress markers of Nicotiana tabacum L. were compared. Chelators (without ZnO) enhanced root growth, whilst ZnO negatively affected seedling growth. ZnO toxicity was often mitigated by adding chelators, especially citrate, although at the highest levels (50 and 100?ppm) of ZnO, toxicity was more pronounced when chelated with EDTA, but was decreased with citrate. Collectively, our findings provide important information regarding the different morpho-physiological and biochemical effects of bulk and nano ZnO and organic and inorganic chelators (citrate and EDTA), which are all prevalent in the environment.     

Second and third harmonic generation microscopy visualizes key structural components in fresh unprocessed healthy human breast tissue

Real‐time assessment of excised tissue may help to improve surgical results in breast tumor surgeries. Here, as a step towards this purpose, the potential of second and third harmonic generation (SHG, THG) microscopy is explored. SHG and THG are nonlinear optical microscopic techniques that do not require labeling of tissue to generate 3D images with intrinsic depth‐sectioning at sub‐cellular resolution. Until now, this technique had been applied on fixated breast tissue or to visualize the stroma only, whereas most tumors start in the lobules and ducts. Here, SHG/THG images of freshly excised unprocessed healthy human tissue are shown to reveal key breast components—lobules, ducts, fat tissue, connective tissue and blood vessels, in good agreement with hematoxylin and eosin histology. DNA staining of fresh unprocessed mouse breast tissue was performed to aid in the identification of cell nuclei in label‐free THG images. Furthermore, 2‐ and 3‐photon excited auto‐fluorescence images of mouse and human tissue are collected for comparison. The SHG/THG imaging modalities generate high quality images of freshly excised tissue in less than a minute with an information content comparable to that of the gold standard, histopathology. Therefore, SHG/THG microscopy is a promising tool for real‐time assessment of excised tissue during surgery.      

Heterozygosity at neutral and immune loci is not associated with neonatal mortality due to microbial infection in Antarctic fur seals

Numerous studies have reported correlations between the heterozygosity of genetic markers and fitness. These heterozygosity–fitness correlations (HFCs) play a central role in evolutionary and conservation biology, yet their mechanistic basis remains open to debate. For example, fitness associations have been widely reported at both neutral and functional loci, yet few studies have directly compared the two, making it difficult to gauge the relative contributions of genome‐wide inbreeding and specific functional genes to fitness. Here, we compared the effects of neutral and immune gene heterozygosity on death from bacterial infection in Antarctic fur seal (Arctocephalus gazella) pups. We specifically developed a panel of 13 microsatellites from expressed immune genes and genotyped these together with 48 neutral loci in 234 individuals, comprising 39 pups that were classified at necropsy as having most likely died of bacterial infection together with a five times larger matched sample of healthy surviving pups. Identity disequilibrium quantified from the neutral markers was positive and significant, indicative of variance in inbreeding within the study population. However, multilocus heterozygosity did not differ significantly between healthy and infected pups at either class of marker, and little evidence was found for fitness associations at individual loci. These results support a previous study of Antarctic fur seals that found no effects of heterozygosity at nine neutral microsatellites on neonatal survival and thereby help to refine our understanding of how HFCs vary across the life cycle. Given that nonsignificant HFCs are underreported in the literature, we also hope that our study will contribute toward a more balanced understanding of the wider importance of this phenomenon.     

Molecular characterization of four Haemophilus influenzae serotype a strains isolated from patients in Quebec, Canada

Four epidemiologically unrelated Haemophilus influenzae serotype a (Hia) strains from patients in Quebec, Canada, were characterized and found to represent 3 distinct groups. One isolate, found to be biotype I and sequence type (ST)-62 by multilocus sequence typing, was shown to possess the copper- and zinc-containing superoxide dismutase gene, sodC, and was suspected to belong to clonal division II. The other 3 isolates were classified as clonal division I based on the absence of the sodC gene. Among the 3 sodC-negative Hia strains, 2 were biotype II and had related STs (ST-23 and ST-403) and highly similar DNA fingerprints, similar to a group of previously described Hia isolates causing invasive disease in Manitoba, Canada. The remaining sodC-negative strain belonged to biotype I and ST-4 and shared no common allele with ST-23, ST-403, or ST-62. This isolate also possessed the IS1016-bexA partial deletion, which is often associated with increased virulence. Despite the small number of isolates used in this study, our finding of 3 distinct groups shows the existence of a potential genetic diversity not previously described for Hia. Whether this genetic diversity is related to the severity and epidemiology of Hia disease requires further studies.     

The Drosophila RNA-binding protein ELAV is required for commissural axon midline crossing via control of commissureless mRNA expression in neurons

Drosophila ELAV is the founding member of an evolutionarily conserved family of RNA-binding proteins considered as key inducers of neuronal differentiation. Although several ELAV-specific targets have been identified, little is known about the role of elav during neural development. Here, we report a detailed characterization of the elav mutant commissural phenotype. The reduced number of commissures in elav mutant embryos is not due to loss or misspecification of neural cells but results from defects in commissural axon projections across the midline. We establish a causal relationship between the elav mutant commissural phenotype and a reduction in the expression of commissureless, a key component of the Robo/Slit growth cone repulsive signalling pathway. In the nerve cord of elav mutant embryos, comm mRNA expression is strongly reduced in neurons, but not in midline glial cells. Furthermore, specific expression of an elav transgene in posterior neurons of each segment of an elav mutant nerve cord restores comm mRNA expression in these cells, as well as the formation of posterior commissures. Finally, forced expression of comm in specific commissural neuron subsets rescues the midline crossing defects of these neurons in elav mutant embryos, further indicating that elav acts cell autonomously on comm expression.     

Structural and functional investigations of Ureaplasma parvum UMP kinase--a potential antibacterial drug target

The crystal structure of uridine monophosphate kinase (UMP kinase, UMPK) from the opportunistic pathogen Ureaplasma parvum was determined and showed similar three-dimensional fold as other bacterial and archaeal UMPKs that all belong to the amino acid kinase family. Recombinant UpUMPK exhibited Michaelis-Menten kinetics with UMP, with K(m) and V(max) values of 214 +/- 4 microm and 262 +/- 24 micromol.min(-1).mg(-1), respectively, but with ATP as variable substrate the kinetic analysis showed positive cooperativity, with an n value of 1.5 +/- 0.1. The end-product UTP was a competitive inhibitor against UMP and a noncompetitive inhibitor towards ATP. Unlike UMPKs from other bacteria, which are activated by GTP, GTP had no detectable effect on UpUMPK activity. An attempt to create a GTP-activated enzyme was made using site-directed mutagenesis. The mutant enzyme F133N (F133 corresponds to the residue in Escherichia coli that is involved in GTP activation), with F133A as a control, were expressed, purified and characterized. Both enzymes exhibited negative cooperativity with UMP, and GTP had no effect on enzyme activity, demonstrating that F133 is involved in subunit interactions but apparently not in GTP activation. The physiological role of UpUMPK in bacterial nucleic acid synthesis and its potential as target for development of antimicrobial agents are discussed.