Mammalian cell production of a respiratory syncytial virus (RSV) candidate vaccine recovered using a product-specific affinity column

The recombinant production of a respiratory syncytial virus (RSV) candidate vaccine BBG2Na in baby hamster kidney cells (BHK-21 cells) was investigated. BBG2Na consists of a serum-albumin-binding region (BB) fused to a 101-amino-acid fragment of the RSV G-protein. Semliki Forest virus-based expression vectors encoding both intracellular and secreted forms of BBG2Na were constructed and found to be functional. Affinity recovery of BBG2Na employing human serum albumin columns was found to be inefficient due to the abundance of BSA in the applied samples. Instead, a strategy using a tailor-made affinity ligand based on a combinatorially engineered Staphylococcus aureus protein A domain, showing specific binding to the G-protein part of the product, was evaluated. In conclusion, a strategy for production and successful recovery of BBG2Na in mammalian cells was created, through the development of a product-specific affinity column.

Impact of cough across different chronic respiratory diseases: comparison of two cough-specific health-related quality of life questionnaires

Background: Cough is a prominent symptom across a range of common chronic respiratory diseases and impacts considerably on patient health status.

Methods: We undertook a cross-sectional comparison of scores from two cough-specific health-related quality of life (HRQoL) questionnaires, the Leicester Cough Questionnaire (LCQ), and the Cough Quality of Life Questionnaire (CQLQ), together with a generic HRQoL measure, the EuroQol. Questionnaires were administered to and spirometry performed on 147 outpatients with chronic cough (n = 83), COPD (n = 18), asthma (n = 20), and bronchiectasis (n = 26).

Results: There was no significant difference in the LCQ and CQLQ total scores between groups (p = 0.24 and p = 0.26, respectively). Exploratory analyses of questionnaire subdomains revealed differences in psychosocial issues and functional impairment between the four groups (p = 0.01 and p = 0.05, respectively). CQLQ scores indicated that chronic coughers have more psychosocial issues than patients with bronchiectasis (p = 0.03) but less functional impairment than COPD patients (p = 0.04). There was a significant difference in generic health status across the four disease groups (p = 0.04), with poorest health status in COPD patients. A significant inverse correlation was observed between CQLQ and LCQ in each disease group (chronic cough r = - 0.56, p < 0.001; COPD r = - 0.49, p = 0.04; asthma r = - 0.94, p < 0.001; and bronchiectasis r = - 0.88, p < 0.001). There was no correlation between cough questionnaire scores and FEV1 in any group, although a significant correlation between EuroQol visual analog scale component and FEV1 (r = 0.639, p = 0.004) was observed in COPD patients.

Conclusion: Cough adversely affects health status across a range of common respiratory diseases. The LCQ and CQLQ can each provide important additional information concerning the impact of cough.

Natural Exposure to Cutaneous Anthrax Gives Long-Lasting T Cell Immunity Encompassing Infection-Specific Epitopes

There has been a long history of defining T cell epitopes to track viral immunity and to design rational vaccines, yet few data of this type exist for bacterial infections. Bacillus anthracis, the causative agent of anthrax, is both an endemic pathogen in many regions and a potential biological warfare threat. T cell immunity in naturally infected anthrax patients has not previously been characterized, which is surprising given concern about the ability of anthrax toxins to subvert or ablate adaptive immunity. We investigated CD4 T cell responses in patients from the Kayseri region of Turkey who were previously infected with cutaneous anthrax. Responses to B. anthracis protective Ag and lethal factor (LF) were investigated at the protein, domain, and epitope level. Several years after antibiotic-treated anthrax infection, strong T cell memory was detectable, with no evidence of the expected impairment in specific immunity. Although serological responses to existing anthrax vaccines focus primarily on protective Ag, the major target of T cell immunity in infected individuals and anthrax-vaccinated donors was LF, notably domain IV. Some of these anthrax epitopes showed broad binding to several HLA class alleles, but others were more constrained in their HLA binding patterns. Of specific CD4 T cell epitopes targeted within LF domain IV, one is preferentially seen in the context of bacterial infection, as opposed to vaccination, suggesting that studies of this type will be important in understanding how the human immune system confronts serious bacterial infection.

Introduction to Domain-Driven Data Mining Special Section

In the last decade, data mining has emerged as one of the most dynamic and lively areas in information technology. Although many algorithms and techniques for data mining have been proposed, they either focus on domain independent techniques or on very specific domain problems. A general requirement in bridging the gap between academia and business is to cater to general domain-related issues surrounding real-life applications, such as constraints, organizational factors, domain expert knowledge, domain adaption, and operational knowledge. Unfortunately, these either have not been addressed, or have not been sufficiently addressed, in current data mining research and development.Domain-Driven Data Mining (D3M) aims to develop general principles, methodologies, and techniques for modeling and merging comprehensive domain-related factors and synthesized ubiquitous intelligence surrounding problem domains with the data mining process, and discovering knowledge to support business decision-making. This paper aims to report original, cutting-edge, and state-of-the-art progress in D3M. It covers theoretical and applied contributions aiming to: 1) propose next-generation data mining frameworks and processes for actionable knowledge discovery, 2) investigate effective (automated, human and machine-centered and/or human-machined-co-operated) principles and approaches for acquiring, representing, modelling, and engaging ubiquitous intelligence in real-world data mining, and 3) develop workable and operational systems balancing technical significance and applications concerns, and converting and delivering actionable knowledge into operational applications rules to seamlessly engage application processes and systems.

A Low Complexity Video Watermarking in H.264 Compressed Domain

In this paper, a new blind and readable H.264 compressed domain watermarking scheme is proposed in which the embedding/extracting is performed using the syntactic elements of the compressed bit stream. As a result, it is not necessary to fully decode a compressed video stream both in the embedding and extracting processes. The method also presents an inexpensive spatiotemporal analysis that selects the appropriate submacroblocks for embedding, increasing watermark robustness while reducing its impact on visual quality. Meanwhile, the proposed method prevents bit-rate increase and restricts it within an acceptable limit by selecting appropriate quantized residuals for watermark insertion. Regarding watermarking demands such as imperceptibility, bit-rate control, and appropriate level of security, a priority matrix is defined which can be adjusted based on the application requirements. The resulted flexibility expands the usability of the proposed method.

Music, Magic, and the Mythic: The Dynamics of Visual and Aural Discourse in Souleymane Cissé’s Yeelen

The 1980s saw a wave of African films that aimed to represent, on both local and international screens, a sophisticated pre-colonial Africa, thus debunking notions of the continent as primitive. Toward this aim the films inscribed the conventions of oral performance within their visual styles, denying spectator identification with the protagonists and emphasising the presence of the narrator. However, some critics argued that these films exoticised Africa, while their use of oral performance’s distancing effect echoed the ‘scientific’ distance structured by the ethnographic film, in which African societies were represented as ‘the other’. Souleymane Cissé’s Yeelen exemplifies this tension, transposing into cinematic form oral storytelling techniques in the depiction of a power struggle within the covert cult of the komo, a Bambara initiation society unfamiliar to most non-Bambara viewers. This paper demonstrates how the film negotiates this tension via music, which interpellates the international spectator by eliciting a greater identification with the protagonists than that determined at a visual level, while encoding a verisimilitude to rituals that may otherwise be read as the superstitious practices of ‘the other’. In this way, music and image in Yeelen operate as parallel, though often overlapping, discourses, bridging the gap between the film’s culturally specific narrative and formal components, and its international spectators.

Induction of endothelial PAS domain protein-1 by hypoxia: Characterization and comparison with hypoxia-inducible factor-1 alpha

Hypoxia results in adaptive changes in the transcription of a range of genes including erythropoietin. An important mediator is hypoxia-inducible factor-1 (HIF-1), a DNA binding complex shown to contain at least two basic helix-loop-helix PAS-domain (bHLH-PAS) proteins, HIF-1 alpha and aryl hydrocarbon nuclear receptor translocator (ARNT), In response to hypoxia, HIF-1 alpha is activated and accumulates rapidly in the cell. Endothelial PAS domain protein 1 (EPAS-1) is a recently identified bHLH-PAS protein with 48% identity to HIF-1 alpha, raising the question of its role in responses to hypoxia. We developed specific antibodies and studied expression and regulation of EPAS-1 mRNA and protein across a range of human cell lines. EPAS-1 was widely expressed, and strongly induced by hypoxia at the level of protein but not mRNA. Comparison of the effect of a range of activating and inhibitory stimuli showed striking similarities in the EPAS-1 and HIF-1 alpha responses. Although major differences were observed in the abundance of EPAS-1 and HIF-1 alpha in different cell types, differences in the inducible response were subtle with EPAS-1 protein being slightly more evident in normoxic and mildly hypoxic cells. Functional studies in a mutant cell line (Ka13) expressing neither HIF-1 alpha nor EPAS-1 confirmed that both proteins interact with hypoxically responsive targets, but suggest target specificity with greater EPAS-1 transactivation (relative to HIF-1 alpha transactivation) of the VEGF promoter than the LDH-A promoter. (C) 1998 by The American Society of Hematology.

Evaluation of a Passive Millimeter Wave (PMMW) imager for wire detection in degraded visual conditions

Ground vehicle tests have been performed to evaluate the performance of a Passive Millimeter Wave (PMMW) imager in reduced visibility conditions and in particular, the ability to detect power lines and cables. A PMMW imager was
compared with Long Wave Infrared (LWIR) and visible imaging cameras. The three sensors were mounted on a Land Rover, together with GPS and digital recording system. All three sensors plus the GPS data were recorded simultaneously in order to provide direct comparisons. The vehicle collected imagery from a number of sites in the vicinity of Malvern, UK, in January, 2008. Imagery was collected both while the vehicle was stationary at specific sites
and while it was moving. Weather conditions during the data collection included clear, drizzle, rain and fog. Imagery was collected during the day, at night, and during dusk/dawn transition periods. The PMMW imager was a prototype which operated at 94 GHz and was based on a conically scanned folded Schmidt camera and the LWIR and visible sensors were commercial off the shelf items.

The Respiratory Syncytial Virus G Protein Conserved Domain Induces a Persistent and Protective Antibody Response in Rodents

Respiratory syncytial virus (RSV) is an important cause of severe upper and lower respiratory disease in infants and in the elderly. There are 2 main RSV subtypes A and B. A recombinant vaccine was designed based on the central domain of the RSV-A attachment G protein which we had previously named G2Na (aa130–230). Here we evaluated immunogenicity, persistence of antibody (Ab) response and protective efficacy induced in rodents by: (i) G2Na fused to DT (Diphtheria toxin) fragments in cotton rats. DT fusion did not potentiate neutralizing Ab responses against RSV-A or cross-reactivity to RSV-B. (ii) G2Nb (aa130–230 of the RSV-B G protein) either fused to, or admixed with G2Na. G2Nb did not induce RSV-B-reactive Ab responses. (iii) G2Na at low doses. Two injections of 3 µg G2Na in Alum were sufficient to induce protective immune responses in mouse lungs, preventing RSV-A and greatly reducing RSV-B infections. In cotton rats, G2Na-induced RSV-reactive Ab and protective immunity against RSV-A challenge that persisted for at least 24 weeks. (iv) injecting RSV primed mice with a single dose of G2Na/Alum or G2Na/PLGA [poly(D,L-lactide-co-glycolide]. Despite the presence of pre-existing RSV-specific Abs, these formulations effectively boosted anti-RSV Ab titres and increased Ab titres persisted for at least 21 weeks. Affinity maturation of these Abs increased from day 28 to day 148. These data indicate that G2Na has potential as a component of an RSV vaccine formulation.

A novel GUCY2D mutation, V933A, causes central areolar choroidal dystrophy

Purpose: To identify the genetic cause of central areolar choroidal dystrophy (CACD) in a large Northern Irish family.
Methods: We previously reported linkage of the locus for CACD in this family to an interval of approximately 5 cM on chromosome 17p13 flanked by polymorphic markers D17S1810 and CHLC GATA7B03. We undertook sequence capture, massively-parallel sequencing and computational alignment, base-calling and annotation to identify a causative mutation. Conventional sequencing was used to confirm the results.
Results: Deep sequencing identified a single-base substitution in guanylate cyclase 2D, membrane (retina-specific) (GUCY2D). The novel mutation segregated with the disease phenotype and resulted in substitution of valine by alanine at position 933, within the catalytic domain of the protein. It altered a motif that is strongly conserved in a large number of distantly related proteins across several species, and was predicted to have a damaging effect on protein activity.
Conclusion: Mutations in GUCY2D have previously been associated with dominant cone rod dystrophies (CORD6) and recessive forms of Leber's congenital amaurosis (LCA). This is the first report of GUCY2D mutation causing CACD and adds to our understanding of genotype-phenotype correlation in this heterogeneous group of choroidoretinal dystrophies.

Conserved amino acid residues found in a predicted cytosolic domain of the lipopolysaccharide biosynthetic protein WecA are implicated in the recognition of UDP-N-acetylglucosamine

WecA, an integral membrane protein that belongs to a family of polyisoprenyl phosphate N-acetylhexosamine-1-phosphate transferases, is required for the biosynthesis of O-specific LPS and enterobacterial common antigen in Escherichia coli and other enteric bacteria. WecA functions as an UDP-N-acetylglucosamine (GlcNAc):undecaprenyl-phosphate GlcNAc-1-phosphate transferase. A conserved short sequence motif (His-Ile-His-His; HIHH) and a conserved arginine were identified in WecA at positions 279-282 and 265, respectively. This region is located within a predicted cytosolic segment common to all bacterial homologues of WecA. Both HIHH279-282 and the Arg265 are reminiscent of the HIGH motif (His-Ile-Gly-His) and a nearby upstream lysine, which contribute to the three-dimensional architecture of the nucleotide-binding site among various enzymes displaying nucleotidyltransferase activity. Thus, it was hypothesized that these residues may play a role in the interaction of WecA with UDP-GlcNAc. Replacement of the entire HIHH motif by site-directed mutagenesis produced a protein that, when expressed in the E. coli wecA mutant MV501, did not complement the synthesis of O7 LPS. Membrane extracts containing the mutated protein failed to transfer UDP-GlcNAc into a lipid-rich fraction and to bind the UDP-GlcNAc analogue tunicamycin. Similar results were obtained by individually replacing the first histidine (H279) of the HIHH motif as well as the Arg265 residue. The functional importance of these residues is underscored by the high level of conservation of H279 and Arg265 among bacterial WecA homologues that utilize several different UDP-N-acetylhexosamine substrates.

Cognitive functioning in subjects with recent-onset psychosis from a low-middle-income environment: Multiple-domain deficits and longitudinal evaluation

Cognitive deficits are a key feature of recent-onset psychosis, but there is no consensus on whether such deficits are generalized or confined to specific domains. Besides, it is unclear whether cognitive deficits: a) are found in psychotic patients in samples from outside high-income countries; and b) whether they progress uniformly over time in schizophrenia and affective psychoses. We applied 12 tests organized into eight cognitive domains, comparing psychosis patients (n = 56, time from initial contact = 677.95+/-183.27 days) versus healthy controls (n = 70) recruited from the same area of Sao Paulo, Brazil. Longitudinal comparisons (digit span and verbal fluency) were conducted between a previous assessment of the subjects carried out at their psychosis onset, and the current follow-up evaluation. Psychosis patients differed significantly from controls on five domains, most prominently on verbal memory. Cognitive deficits remained detectable in separate comparisons of the schizophrenia subgroup and, to a lesser extent, the affective psychosis subjects against controls. Longitudinal comparisons indicated significant improvement in schizophrenia, affective psychoses, and control subjects, with no significant group-by-time interactions. Our results reinforce the view that there are generalized cognitive deficits in association with recent-onset psychoses, particularly of non-affective nature, which persist over time. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Robust Audio-Visual Speech Recognition under Noisy Audio-Video Conditions

This paper presents the maximum weighted stream posterior (MWSP) model as a robust and efficient stream integration method for audio-visual speech recognition in environments, where the audio or video streams may be subjected to unknown and time-varying corruption. A significant advantage of MWSP is that it does not require any specific measurements of the signal in either stream to calculate appropriate stream weights during recognition, and as such it is modality-independent. This also means that MWSP complements and can be used alongside many of the other approaches that have been proposed in the literature for this problem. For evaluation we used the large XM2VTS database for speaker-independent audio-visual speech recognition. The extensive tests include both clean and corrupted utterances with corruption added in either/both the video and audio streams using a variety of types (e.g., MPEG-4 video compression) and levels of noise. The experiments show that this approach gives excellent performance in comparison to another well-known dynamic stream weighting approach and also compared to any fixed-weighted integration approach in both clean conditions or when noise is added to either stream. Furthermore, our experiments show that the MWSP approach dynamically selects suitable integration weights on a frame-by-frame basis according to the level of noise in the streams and also according to the naturally fluctuating relative reliability of the modalities even in clean conditions. The MWSP approach is shown to maintain robust recognition performance in all tested conditions, while requiring no prior knowledge about the type or level of noise.