mRNA Display Design of Fibronectin-based Intrabodies That Detect and Inhibit Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein

The nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) coronavirus plays important roles in both viral replication and modulation of host cell processes. New ligands that target the N protein may thus provide tools to track the protein inside cells, detect interaction hot spots on the protein surface, and discover sites that could be used to develop new anti-SARS therapies. Using mRNA display selection and directed evolution, we designed novel antibody-like protein affinity reagents that target SARS N protein with high affinity and selectivity. Our libraries were based on an 88-residue variant of the 10th fibronectin type III domain from human fibronectin (10Fn3). This selection resulted in eight independent 10Fn3 intrabodies, two that require the N-terminal domain for binding and six that recognize the C terminus, one with K_d = 1.7 nM. 10Fn3 intrabodies are well expressed in mammalian cells and are relocalized by N in SARS-infected cells. Seven of the selected intrabodies tested do not perturb cellular function when expressed singly in vivo and inhibit virus replication from 11- to 5900-fold when expressed in cells prior to infection. Targeting two sites on SARS-N simultaneously using two distinct 10Fn3s results in synergistic inhibition of virus replication

Consenting to health record linkage: evidence from a multi-purpose longitudinal survey of a general population

Background: The British Household Panel Survey (BHPS) is the first long-running UK longitudinal survey with a non-medical focus and a sample covering the whole age range to have asked for permission to link to a range of administrative health records. This study determines whether informed consent led to selection bias and reflects on the value of the BHPS linked with health records for epidemiological research. Methods. Multivariate logistical regression is used, with whether the respondent gave consent to data linkage or not as the dependent variable. Independent variables were entered as four blocks; (i) a set of standard demographics likely to be found in most health registration data, (ii) a broader set of socio-economic characteristics, (iii) a set of indicators of health conditions and (iv) information about the use of health services. Results: Participants aged 16-24, males and those living in England were more likely to consent. Consent is not biased with respect to socio-economic characteristics or health. Recent users of GP services are underrepresented among consenters. Conclusions: Whilst data could only be linked for a minority of BHPS participants, the BHPS offers a great range of information on people's life histories, their attitudes and behaviours making it an invaluable source for epidemiological research. © 2012 Knies et al; licensee BioMed Central Ltd

Pupil dilation to illusory motion in peripheral drift images: Perception versus reality

Peripheral drift is a specific type of illusory motion that causes observers to perceive motion in a static image. We aimed to determine whether pupil dilation occurs during the perception of illusory motion. In three experiments investigating pupil-size changes to peripheral drift, pupil response differences were observed between symmetric patterns (SPs) that elicited no impression of motion and repeated asymmetric patterns (RAPs) that did. All participants reported the perception of motion in the RAP condition and showed significantly greater pupil dilation to these stimuli as compared with viewing stimuli in the SP condition. As a follow-up, we manipulated the RAP stimuli to reduce and then remove the illusion to determine (a) whether it was the asymmetry per se that induced the pupil dilation and (b) whether the amount of pupil dilation was contingent on the amount of observed illusory motion. Although a reduction in perceived illusory motion did not produce a reduction in pupil dilation, removal of the illusory motion did. Despite previous evidence reporting pupil constriction to the perception of motion, and the positive valence associated with symmetry, these experiments show that pupil dilation occurs during the perception of illusory motion. This is in keeping with previous evidence that pupil dilation is influenced by perceptual factors and not simply light level, and, in particular, shows that illusory motion is physiologically arousingCanadian Institute of Healt

Winter wheat roots grow twice as deep as spring wheat roots, is this important for N uptake and N leaching losses?

Cropping systems comprising winter catch crops followed by spring wheat could reduce N leaching risks compared to traditional winter wheat systems in humid climates. We studied the soil mineral N (Ninorg) and root growth of winter- and spring wheat to 2.5 m depth during three years. Root depth of winter wheat (2.2 m) was twice that of spring wheat, and this was related to much lower amounts of Ninorg in the 1 to 2.5 m layer after winter wheat (81 kg Ninorg ha-1 less). When growing winter catch crops before spring wheat, N content in the 1 to 2.5 m layer after spring wheat was not different from that after winter wheat. The results suggest that by virtue of its deep rooting, winter wheat may not lead to high levels of leaching as it is often assumed in humid climates. Deep soil and root measurements (below 1 m) in this experiment were essential to answer the questions we posed

Activity of the DNA minor groove cross-linking agent SG2000 (SJG-136) against canine tumours

BACKGROUND: Cancer is the leading cause of death in older dogs and its prevalence is increasing. There is clearly a need to develop more effective anti-cancer drugs in dogs. SG2000 (SJG-136) is a sequence selective DNA minor groove cross-linking agent. Based on its in vitro potency, the spectrum of in vivo and clinical activity against human tumours, and its tolerability in human patients, SG2000 has potential as a novel therapeutic against spontaneously occurring canine malignancies. RESULTS: In vitro cytotoxicity was assessed using SRB and MTT assays, and in vivo activity was assessed using canine tumour xenografts. DNA interstrand cross-linking (ICL) was determined using a modification of the single cell gel electrophoresis (comet) assay. Effects on cell cycle distribution were assessed by flow cytometry and measurement of γ-H2AX by immunofluorescence and immunohistochemistry. SG2000 had a multi-log differential cytotoxic profile against a panel of 12 canine tumour cell lines representing a range of common tumour types in dogs. In the CMeC-1 melanoma cell line, DNA ICLs increased linearly with dose following a 1 h treatment. Peak ICL was achieved within 1 h and no removal was observed over 48 h. A relationship between DNA ICL formation and cytotoxicity was observed across cell lines. The formation of γ-H2AX foci was slow, becoming evident after 4 h and reaching a peak at 24 h. SG2000 exhibited significant anti-tumour activity against two canine melanoma tumour models in vivo. Anti-tumour activity was observed at 0.15 and 0.3 mg/kg given i.v. either once, or weekly x 3. Dose-dependent DNA ICL was observed in tumours (and to a lower level in peripheral blood mononuclear cells) at 2 h and persisted at 24 h. ICL increased following the second and third doses in a repeated dose schedule. At 24 h, dose dependent γ-H2AX foci were more numerous than at 2 h, and greater in tumours than in peripheral blood mononuclear cells. SG2000-induced H2AX phosphorylation measured by immunohistochemistry showed good correspondence, but less sensitivity, than measurement of foci. CONCLUSIONS: SG2000 displayed potent activity in vitro against canine cancer cell lines as a result of the formation and persistence of DNA ICLs. SG2000 also had significant in vivo antitumour activity against canine melanoma xenografts, and the comet and γ-H2AX foci methods were relevant pharmacodynamic assays. The clinical testing of SG2000 against spontaneous canine cancer is warranted. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12917-015-0534-2) contains supplementary material, which is available to authorized users

Phage-mediated horizontal transfer of a Staphylococcus aureus virulence-associated genomic island

Staphylococcus aureus is a major pathogen of humans and animals. The capacity of S. aureus to adapt to different host species and tissue types is strongly influenced by the acquisition of mobile genetic elements encoding determinants involved in niche adaptation. The genomic islands νSaα and νSaβ are found in almost all S. aureus strains and are characterized by extensive variation in virulence gene content. However the basis for the diversity and the mechanism underlying mobilization of the genomic islands between strains are unexplained. Here, we demonstrated that the genomic island, νSaβ, encoding an array of virulence factors including staphylococcal superantigens, proteases, and leukotoxins, in addition to bacteriocins, was transferrable in vitro to human and animal strains of multiple S. aureus clones via a resident prophage. The transfer of the νSaβ appears to have been accomplished by multiple conversions of transducing phage particles carrying overlapping segments of the νSaβ. Our findings solve a long-standing mystery regarding the diversification and spread of the genomic island νSaβ, highlighting the central role of bacteriophages in the pathogenic evolution of S. aureus

Acute isolated acetabular fracture following a game of squash: a case report

Although hip injuries do not account a large amount of the Sports Physician's workload they can result in significant morbidity. We present a case where an acetabular fracture was sustained in a relatively young female while playing squash without any history of fall or injury but was treated successfully non-operatively. Such patients who present with acute hip pain must not be dismissed as simply having a soft tissue injury

Inductively guided circuits for ultracold dressed atoms

Recent progress in optics, atomic physics and material science has paved the way to study quantum effects in ultracold atomic alkali gases confined to non-trivial geometries. Multiply connected traps for cold atoms can be prepared by combining inhomogeneous distributions of DC and radio-frequency electromagnetic fields with optical fields that require complex systems for frequency control and stabilization. Here we propose a flexible and robust scheme that creates closed quasi-one-dimensional guides for ultracold atoms through the ‘dressing’ of hyperfine sublevels of the atomic ground state, where the dressing field is spatially modulated by inductive effects over a micro-engineered conducting loop. Remarkably, for commonly used atomic species (for example, 7Li and 87Rb), the guide operation relies entirely on controlling static and low-frequency fields in the regimes of radio-frequency and microwave frequencies. This novel trapping scheme can be implemented with current technology for micro-fabrication and electronic control

Modulation of topoisomerase IIα expression and chemosensitivity through targeted inhibition of NF-Y:DNA binding by a diamino p-anisyl-benzimidazole (Hx) polyamide

BACKGROUND: Sequence specific polyamide HxIP 1, targeted to the inverted CCAAT Box 2 (ICB2) on the topoisomerase IIα (topo IIα) promoter can inhibit NF-Y binding, re-induce gene expression and increase sensitivity to etoposide. To enhance biological activity, diamino-containing derivatives (HxI*P 2 and HxIP* 3) were synthesised incorporating an alkyl amino group at the N1-heterocyclic position of the imidazole/pyrrole. METHODS: DNase I footprinting was used to evaluate DNA binding of the diamino Hx-polyamides, and their ability to disrupt the NF-Y:ICB2 interaction assessed using EMSAs. Topo IIα mRNA (RT-PCR) and protein (Immunoblotting) levels were measured following 18h polyamide treatment of confluent A549 cells. γH2AX was used as a marker for etoposide-induced DNA damage after pre-treatment with HxIP* 3 and cell viability was measured using Cell-Titer Glo®. RESULTS: Introduction of the N1-alkyl amino group reduced selectivity for the target sequence 5'-TACGAT-3' on the topo IIα promoter, but increased DNA binding affinity. Confocal microscopy revealed both fluorescent diamino polyamides localised in the nucleus, yet HxI*P 2 was unable to disrupt the NF-Y:ICB2 interaction and showed no effect against the downregulation of topo IIα. In contrast, inhibition of NF-Y binding by HxIP* 3 stimulated dose-dependent (0.1-2μM) re-induction of topo IIα and potentiated cytotoxicity of topo II poisons by enhancing DNA damage. CONCLUSIONS: Polyamide functionalisation at the N1-position offers a design strategy to improve drug-like properties. Dicationic HxIP* 3 increased topo IIα expression and chemosensitivity to topo II-targeting agents. GENERAL SIGNIFICANCE: Pharmacological modulation of topo IIα expression has the potential to enhance cellular sensitivity to clinically-used anticancer therapeutics. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani