Isolation and characterization of thirty microsatellite loci for Atlantic mackerel (Scomber scombrus L.)

Atlantic mackerel (Scomber scombrus L.) is a widely distributed commercially important pelagic species. Little is known about the stock structure of this species, but it is thought to be undergoing a range extension due to environmental changes. Knowledge of the stock structure under these changing conditions is fundamental for effective management. In this paper, 30 highly polymorphic microsatellite loci developed with next generation sequencing are described. The number of alleles per locus ranged from 4 to 39 in two geographically distant populations, observed and expected heterozygosities ranged between 0. 370-0. 978 and 0. 426-0. 962, respectively. These loci are an important resource that will allow assessment of the current population genetic structure of this species, and enable monitoring of climate related changes in the species range and distribution. 

The discrepancy between social isolation and loneliness as a clinically meaningful metric: findings from the Irish and English longitudinal studies of ageing (TILDA and ELSA)

Objective
Scant evidence is available on the discordance between loneliness and social isolation among older adults. We aimed to investigate this discordance and any health implications that it may have.

Method
Using nationally representative datasets from ageing cohorts in Ireland (TILDA) and England (ELSA), we created a metric of discordance between loneliness and social isolation, to which we refer as Social Asymmetry. This metric was the categorised difference between standardised scores on a scale of loneliness and a scale of social isolation, giving categories of: Concordantly Lonely and Isolated, Discordant: Robust to Loneliness, or Discordant: Susceptible to Loneliness. We used regression and multilevel modelling to identify potential relationships between Social Asymmetry and cognitive outcomes.

Results
Social Asymmetry predicted cognitive outcomes cross-sectionally and at a two-year follow-up, such that Discordant: Robust to Loneliness individuals were superior performers, but we failed to find evidence for Social Asymmetry as a predictor of cognitive trajectory over time.

Conclusions
We present a new metric and preliminary evidence of a relationship with clinical outcomes. Further research validating this metric in different populations, and evaluating its relationship with other outcomes, is warranted.

Isolation and structural determination of non-racemic tertiary cathinone derivatives

The racemic tertiary cathinones N,N-dimethylcathinone (1), N,N-diethylcathinone (2) and 2-(1-pyrrolidinyl)-propiophenone (3) have been prepared in reasonable yield and characterized using NMR and mass spectroscopy. HPLC indicates that these compounds are isolated as the anticipated racemic mixture. These can then be co-crystallized with (+)-O,O′-di-p-toluoyl-d-tartaric, (+)-O,O′-dibenzoyl-d-tartaric and (-)-O,O′-dibenzoyl-l-tartaric acids giving the single enantiomers S and R respectively of 1, 2 and 3, in the presence of sodium hydroxide through a dynamic kinetic resolution. X-ray structural determination confirmed the enantioselectivity. The free amines could be obtained following basification and extraction. In methanol these are reasonably stable for the period of several hours, and their identity was confirmed by HPLC and CD spectroscopy.

Broadband Dual-Polarized Antenna with High Port Isolation and Polarization Purity

This paper presents a novel high symmetry balun which significantly improves the performance of dipole-based dual-polarized antennas. The new balun structure provides enhanced differential capability leading to high performance in terms of port-to-port isolation and far-field cross polarization. An example antenna using this balun is proposed. The simulated results show 53.5% of fractional bandwidth within the band 1.71−2.96 GHz (VSWR<1.5) and port-to-port isolation >59 dB. The radiation characteristic shows around 9 dBi of gain and far-field cross polarization <−48 dBi over the entire bandwidth. The detailed balun functioning and full antenna measurements will be presented during the conference. Performance comparison with similar structures will be also provided.

Data for the co-expression and purification of human recombinant CaMKK2 in complex with calmodulin in Escherichia coli

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in a range of conditions and pathologies from prostate to hepatic cancer. Here, we describe the expression in Escherichia coli and the purification protocol for the following constructs: full-length CaMKK2 in complex with CaM, CaMKK2 'apo', CaMKK2 (165-501) in complex with CaM, and the CaMKK2 F267G mutant. The protocols described have been optimized for maximum yield and purity with minimal purification steps required and the proteins subsequently used to develop a fluorescence-based assay for drug binding to the kinase, "Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2"

Isolation of scorpion (Androctonus amoreuxi) alpha-neurotoxins and parallel cloning of their respective cDNAs from a single sample of venom

The venoms of buthid scorpions are known to contain basic, single-chain protein toxins (alpha toxins) consisting of 60–70 amino acid residues that are tightly folded by four disulfide bridges. Here we describe isolation and sequencing of three novel putative alpha toxins (AamH1-3) from the venom of the North African scorpion, Androctonus amoreuxi, and subsequent cloning of their precursor cDNAs from the same sample of venom. This experimental approach can expedite functional genomic analyses of the protein toxins from this group of venomous animals and does not require specimen sacrifice for cloning of protein toxin precursor cDNAs.

NEUROPEPTIDE-Y AND NEUROPEPTIDE-Y 3-36 - ISOLATION FROM HUMAN PANCREATIC ENDOCRINE TUMORS

Using an antiserum raised to the C-terminal region of neuropeptide Y (NPY) which does not cross-react with pancreatic polypeptide (PP), immunoreactivity has been detected in two different endocrine tumours of the human pancreas in concentrations permitting isolation and structural analysis. In a clinically-typical gastrinoma, resected from the head of pancreas, the concentration of NPY immunoreactivity was 3.4 nmol/g. Reverse phase HPLC analysis of extracts of this tumour resolved a single immunoreactive peptide coeluting with synthetic human NPY. The molecular mass of the isolated peptide, determined by mass spectroscopy, was 4270 Da, which was in close agreement with that derived from the deduced primary structure of human tumour NPY (4271.7 Da), obtained by gas-phase sequencing. A somatostatinoma, resected from the region of the ampulla of Vater, contained 3.8 nmol/g of NPY immunoreactivity and isolation of this immunoreactive peptide followed by structural analyses, indicated a molecular structure consistent with NPY 3-36. These data suggest that NPY immunoreactivity detected in human pancreatic endocrine tumours is molecularly heterogenous, a finding which may be of relevance in the symptomatology of such tumours as attenuation of the N-terminus of this peptide generates receptor selectivity.

Culture and Characterization of Microglia from the Adult Murine Retina

Purpose. To develop a protocol for isolating and culturing murine adult retinal microglia and to characterize the phenotype and function of the cultured cells. Method. Retinal single-cell suspensions were prepared from adult MF1 mice. Culture conditions including culture medium, growth factors, seeding cell density, and purification of microglia from the mixed cultures were optimised. Cultured retinal microglial cells were phenotyped using the surface markers CD45, CD11b, and F4/80. Their ability to secrete proinflammatory cytokines in response to lipopolysaccharide (LPS) stimulation was examined using cytometric bead array (CBA) assay. Results. Higher yield was obtained when retinal single-cell suspension was cultured at the density of cells per cm2 in Dulbecco’s modified Eagle medium (DMEM)/F12 + Glutamax supplement with 20% fetal calf serum (FCS) and 20% L929 supernatant. We identified day 10 to be the optimum day of microglial isolation. Over 98% of the cells isolated were positive for CD45, CD11b, and F4/80. After stimulating with LPS they were able to secrete proinflammatory cytokines such as IL-6 and TNF-α and express CD86, CD40, and MHC-II. Conclusion. We have developed a simple method for isolating and culturing retinal microglia from adult mice.

Purification of a multidrug resistance transporter for crystallization studies

Crystallization of integral membrane proteins is a challenging field and much effort has been invested in optimizing the overexpression and purification steps needed to obtain milligram amounts of pure, stable, monodisperse protein sample for crystallography studies. Our current work involves the structural and functional characterization of the Escherichia coli multidrug resistance transporter MdtM, a member of the major facilitator superfamily (MFS). Here we present a protocol for isolation of MdtM to increase yields of recombinant protein to the milligram quantities necessary for pursuit of structural studies using X-ray crystallography. Purification of MdtM was enhanced by introduction of an elongated His-tag, followed by identification and subsequent removal of chaperonin contamination. For crystallization trials of MdtM, detergent screening using size exclusion chromatography determined that decylmaltoside (DM) was the shortest-chain detergent that maintained the protein in a stable, monodispersed state. Crystallization trials of MdtM performed using the hanging-drop diffusion method with commercially available crystallization screens yielded 3D protein crystals under several different conditions. We contend that the purification protocol described here may be employed for production of high-quality protein of other multidrug efflux members of the MFS, a ubiquitous, physiologically and clinically important class of membrane transporters.

Structural and functional analogs of the novel mammalian neuropeptide, neuromedin S (NmS), in the dermal venoms of Eurasian bombinid toads.

We report the isolation and structural characterization of two neuromedin S (NmS) analogs, (NmS-17 and NmS-33), from the dermal venoms of Eurasian bombinid toads. NmS is a novel neuromedin U (NmU)-related peptide with potent anorexigenic and circadian rhythm-modulating properties recently discovered in mammals. Cloning of NmS precursor-encoding cDNAs from skin venom-derived libraries revealed the presence of a high degree of transcript splice variation comparable to that found previously for NmU in both amphibian skin and mammalian brain. Synthetic replicates of both amphibian NmS peptides evoked robust and dose-dependent transient increases in intracellular calcium ion concentrations in CHO cells that had been stably transfected with either FM-3/GPR66 or FM-4/TGR-1 human NmU receptors. The potency and efficacy of these amphibian skin peptides at such receptors were comparable to those observed with human NmS and rat NmS. These data show that NmS and NmU genes had already diverged at the level of the Amphibia and that differential splicing of their transcribed mRNAs has been highly conserved throughout tetrapod vertebrate evolution indicative of fundamental biological function. NmS is additionally a novel neuropeptide homolog that can be added to the biologically active peptide arsenal of amphibian venom/defensive skin secretions.