Generation of high-affinity chicken single-chain Fv antibody fragments for measurement of the Pseudonitzschia pungens toxin domoic acid

Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single V(H) and V(L) genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules.     

Sleep quality,sleepiness and the influence of workplace breaks: A cross-sectional survey of health-care workers in two US hospitals

ABSTRACT

This study assessed sleep quality, sleepiness and use of workplace break opportunities in 1285 health-care workers via an online questionnaire. Two hospitals were surveyed – one with and one without a fatigue mitigation policy. Across all respondents, 68.9% reported generally taking breaks of at least 30 min and 21.7% had access to a quiet place to rest, with no significant differences between hospitals. The presence of a fatigue mitigation policy was not associated with reduced sleepiness. However, accounting for hospital and shift characteristics, employees with access to a quiet place to rest while on break had significantly lower self-reported sleepiness scores.     

The Diversity of Yellow-Related Proteins in Sand Flies (Diptera: Psychodidae)

Yellow-related proteins (YRPs) present in sand fly saliva act as affinity binders of bioamines, and help the fly to complete a bloodmeal by scavenging the physiological signals of damaged cells. They are also the main antigens in sand fly saliva and their recombinant form is used as a marker of host exposure to sand flies. Moreover, several salivary proteins and plasmids coding these proteins induce strong immune response in hosts bitten by sand flies and are being used to design protecting vaccines against Leishmania parasites. In this study, thirty two 3D models of different yellow-related proteins from thirteen sand fly species of two genera were constructed based on the known protein structure from Lutzomyia longipalpis. We also studied evolutionary relationships among species based on protein sequences as well as sequence and structural variability of their ligand-binding site. All of these 33 sand fly YRPs shared a similar structure, including a unique tunnel that connects the ligand-binding site with the solvent by two independent paths. However, intraspecific modifications found among these proteins affects the charges of the entrances to the tunnel, the length of the tunnel and its hydrophobicity. We suggest that these structural and sequential differences influence the ligand-binding abilities of these proteins and provide sand flies with a greater number of YRP paralogs with more nuanced answers to bioamines. All these characteristics allow us to better evaluate these proteins with respect to their potential use as part of anti-Leishmania vaccines or as an antigen to measure host exposure to sand flies.     

Nigrostriatal dopaminergic deficits and hypokinesia caused by inactivation of the familial Parkinsonism-linked gene DJ-1

The manifestations of Parkinson's disease are caused by reduced dopaminergic innervation of the striatum. Loss-of-function mutations in the DJ-1 gene cause early-onset familial parkinsonism. To investigate a possible role for DJ-1 in the dopaminergic system, we generated a mouse model bearing a germline disruption of DJ-1. Although DJ-1(-/-) mice had normal numbers of dopaminergic neurons in the substantia nigra, evoked dopamine overflow in the striatum was markedly reduced, primarily as a result of increased reuptake. Nigral neurons lacking DJ-1 were less sensitive to the inhibitory effects of D2 autoreceptor stimulation. Corticostriatal long-term potentiation was normal in medium spiny neurons of DJ-1(-/-) mice, but long-term depression (LTD) was absent. The LTD deficit was reversed by treatment with D2 but not D1 receptor agonists. Furthermore, DJ-1(-/-) mice displayed hypoactivity in the open field. Collectively, our findings suggest an essential role for DJ-1 in dopaminergic physiology and D2 receptor-mediated functions.     

Differences in rhizome aeration of Phragmites australis in a constructed wetland

Waterlogged soils are a challenging environment for plants due to anoxic conditions and enrichment of phytotoxic substances in the rhizosphere. High contents of organic matter in the substrate as present in constructed wetlands (CW) may amplify these effects. The influence of substrate organic matter on the rhizome aeration of Phragmites australis (common reed) was investigated in a CW for sewage treatment on two sites with different organic concentrations: (a) inflow part of the bed with a high organic charge (high-organic site) and (b) outflow zone of the bed with a lower organic charge (low-organic site). Several diurnal oxygen (O₂) courses were recorded inside the rhizomes using micro-optodes. Maximum O₂ percentages in the rhizomes were generally the same at both sites, but minimum O₂ values were lower in the low-organic site than in the high-organic site, leading to higher amplitudes at the low-organic site. The results suggest that oxygen release from the roots is hampered under high-organic compared to low-organic conditions. This observation might be explained by changes in gas conductivity of the roots, but also by alterations in microbial oxygen demand, under different organic burden.     

Huntingtin fragments that aggregate go their separate ways

N-terminal region of mutant huntingtin forms intranuclear and cytoplasmic aggregates in neurons that may contribute to neuronal death in Huntington's disease. show that different endoprotease-cleaved huntingtin fragments form nuclear and cytoplasmic inclusions.     

Membrane Transport Generated by the Osmotic and Hydrostatic Pressure. Correlation Relation for Parameters L p, σ, and ω

Standard approach to membrane transport generated by osmotic andhydrostatic pressures, developed by Kedem and Katchalsky, is based onprinciples of thermodynamics of irreversible processes. In this paper wepropose an alternative technique. We derive transport equations from fewfairly natural assumptions and a mechanistic interpretation of the flows.In particular we postulate that a sieve-type membrane permeability isdetermined by the pore sizes and these are random within certain range.Assuming that an individual pore is either permeable or impermeable tosolute molecules, the membrane reflection coefficient depends on the ratioof permeable and impermeable pores. Considering flows through permeableand impermeable pores separately, we derive equations for the total volumeflux, solute flux and the solvent flux across the membrane. Comparing themechanistic equations to the Kedem-Katchalsky equations we find the formereasier to interpret physically. Based on the mechanistic equations we alsoderive a correlation relation for the membrane transport parameters L _p,, and . This relation eliminates the need for experimentaldetermination of all three phenomenological parameters, which in somecases met with considerable difficulties.     

Isolation of Circadian Clock Mutants of NEUROSPORA CRASSA

Three mutants of Neurospora crassa have been isolated which have altered period lengths of their circadian rhythm of conidiation. The strains, designated "frequency" (frq), were obtained after mutagenesis of the band (bd) strain with N-methyl-N'-nitro-N-nitrosoguanidine. In continuous darkness at 25 degrees bd has a period length of 21.6 +/- 0.5 hours; under the same conditions the period length of frq-1 is 16.5 +/- 0.5 hours; frq-2, 19.3 +/- 0.4 hours; and frq-3, 24.0 +/- 0.4 hours. Each of the mutants segregates as a single nuclear gene. All three mutants appear very tightly linked to each other, but it has not yet been determined whether the mutants are allelic. No major changes in the responses to light and temperature have been observed in any of the mutants. It is suggested that these mutants represent alterations in the basic timing mechanism of the circadian clock of Neurospora.