A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control

BACKGROUND: Avian influenza viruses (AIVs) are endemic in wild birds and their introduction and conversion to highly pathogenic avian influenza virus in domestic poultry is a cause of serious economic losses as well as a risk for potential transmission to humans. The ability to rapidly recognise AIVs in biological specimens is critical for limiting further spread of the disease in poultry. The advent of molecular methods such as real time polymerase chain reaction has allowed improvement of detection methods currently used in laboratories, although not all of these methods include an Internal Positive Control (IPC) to monitor for false negative results. Therefore we developed a one-step reverse transcription real time PCR (RRT-PCR) with a Minor Groove Binder (MGB) probe for the detection of different subtypes of AIVs. This technique also includes an IPC. METHODS: RRT-PCR was developed using an improved TaqMan technology with a MGB probe to detect AI from reference viruses. Primers and probe were designed based on the matrix gene sequences from most animal and human A influenza virus subtypes. The specificity of RRT-PCR was assessed by detecting influenza A virus isolates belonging to subtypes from H1–H13 isolated in avian, human, swine and equine hosts. The analytical sensitivity of the RRT-PCR assay was determined using serial dilutions of in vitro transcribed matrix gene RNA. The use of a rodent RNA as an IPC in order not to reduce the efficiency of the assay was adopted. RESULTS: The RRT-PCR assay is capable to detect all tested influenza A viruses. The detection limit of the assay was shown to be between 5 and 50 RNA copies per reaction and the standard curve demonstrated a linear range from 5 to 5 × 10(8 )copies as well as excellent reproducibility. The analytical sensitivity of the assay is 10–100 times higher than conventional RT-PCR. CONCLUSION: The high sensitivity, rapidity, reproducibility and specificity of the AIV RRT-PCR with the use of IPC to monitor for false negative results can make this method suitable for diagnosis and for the evaluation of viral load in field specimens

Photosynthetic activity in both algae and cyanobacteria changes in response to cues of predation

A plethora of adaptive responses to predation has been described in microscopic aquatic producers. Although the energetic costs of these responses are expected, with their consequences going far beyond an individual, their underlying molecular and metabolic mechanisms are not fully known. One, so far hardly considered, is if and how the photosynthetic efficiency of phytoplankton might change in response to the predation cues. Our main aim was to identify such responses in phytoplankton and to detect if they are taxon-specific. We exposed seven algae and seven cyanobacteria species to the chemical cues of an efficient consumer, Daphnia magna, which was fed either a green alga, Acutodesmus obliquus, or a cyanobacterium, Synechococcus elongatus (kairomone and alarm cues), or was not fed (kairomone alone). In most algal and cyanobacterial species studied, the quantum yield of photosystem II increased in response to predator fed cyanobacterium, whereas in most of these species the yield did not change in response to predator fed alga. Also, cyanobacteria tended not to respond to a non-feeding predator. The modal qualitative responses of the electron transport rate were similar to those of the quantum yield. To our best knowledge, the results presented here are the broadest scan of photosystem II responses in the predation context so far

Using pretreatment and posttreatment assessments to enhance and evaluate existing treatment packages.

Pretreatment assessment data were used to enhance an existing treatment package to reduce aggression and to increase positive social interactions between a young boy and his peers. Based on the results of pretreatment assessments, additional reinforcement (differential reinforcement of alternative behavior with adult attention) and punishment (performing a nonpreferred task during time-out) components were added to an existing nonresetting differential reinforcement of other behavior (access to peers unless aggression occurred) plus time-out procedure. A posttreatment component analysis of the additional treatment components indicated that the reinforcement component facilitated positive social interactions and the punishment component suppressed aggression towards peers

Wearable Sensors for Frequency-Multiplexed EIT and Multilead ECG Data Acquisition

This paper presents a wearable sensor architecture for frequency-multiplexed electrical impedance tomography (EIT) and synchronous multilead electrocardiogram (ECG) data acquisition. The system is based on a novel electronic sensing architecture, called cooperative sensors, that significantly reduces the cabling complexity and enables flexible EIT stimulation and measurement patterns. The cooperative-sensor architecture was initially designed for ECG and has been extended for multichannel bioimpedance measurement. This approach allows for an adjustable EIT stimulation pattern via frequency-division multiplexing. This paper also shows a calibration procedure as well as EIT system noise performance assessment. Preliminary measurements on a healthy volunteer showed the ability of the wearable system to measure EIT data synchronously with multilead ECG. Ventilation-related and heartbeat-related EIT images were reconstructed, demonstrating the feasibility of the proposed architecture for non-invasive cardiovascular monitoring

Training students with profound or multiple handicaps to make requests via microswitches.

In a series of three experiments, we evaluated the use of microswitches as a means for students with profound, multiple handicaps to demonstrate preferences between toys and to make requests for specific activities. In Experiment 1, 5 students learned to demonstrate toy preferences by using microswitches to activate battery-operated toys. Experiment 2 was conducted to evaluate the students' preferences for social attention. Microswitches were used to activate prerecorded messages that signaled the classroom teacher to attend to the students. In Experiment 3, the students used the switches and prerecorded messages to make specific requests of educational staff in school and community settings. Results of these experiments, evaluated within multiple baseline, alternating treatments, and simultaneous treatments designs, indicated that these students could request specific activities. Results are discussed with respect to the continued use of microswitches and to program development

Sequence alignments for the CH1 region of normalized apoptosis induction potential of Rituximab CH1 variants.

<p>(A) Two parts of the CH1 region, from residues 131 to 138 and 192 to 214, are illustrated. Sequences differences between the control IgG1, IgG2, IgG4P and all engineered CH1 mutants are shaded. (B) The bars illustrate the normalized apoptosis values observed for all Rituximab CH1 variants in relation to the IgG1 control (100%, dashed line) in the absence (black bars) and presence (white bars) of crosslinking antibody. Values for full length IgG2 and IgG4P are shown as a reference. Shown are the means of at least three independent assays ± standard deviation. ** p ≤ 0.01. The p values that are shown were calculated by one way ANOVA using IgG1 in absence of the crosslinker as the control group.</p

Normalized apoptosis induction potential of Rituximab variants combining hinge and CH1 mutations and mutants covering all possible amino acid exchanges of hinge position 219.

<p>(A) The bars illustrate the normalized apoptosis values observed for Rituximab variants that combine hinge and CH1 mutations. Values for full length IgG2 and IgG4P are shown as a reference. (B) shows the impact of all possible amino acid exchanges at hinge position S219. All values are shown in relation to the IgG1 control (100%, dashed line) in the absence (black bars) and presence (white bars) of crosslinking antibody. Data represents the means of at least three independent assays ± standard deviation. * p ≤ 0.05; ** p ≤ 0.01. The shown p values were calculated by one way ANOVA using IgG1 in absence of the crosslinker as the control group.</p

Binding of RTX variants to the surface of Ramos cells and mediation of ADCC via RTX and NKL cells <i>in vitro</i>.

<p>In (A) the binding of RTX variants to the surface of Ramos cells measured by flow cytometry is shown. Two RTX amounts (1μg, white bars) and 0.5μg (black bars) per 1E6 Ramos cells were used. The IgG1 signal ratio represents the MFI of the RTX variant divided by the MFI of the IgG1 control. (B) shows the result of a RTX specific ADCC assay in which three different concentrations (125nM, 31.3nM, 7.8nM) of the antibodies were incubated with Ramos target and NKL effector cells to evaluate ADCC activity of the RTX variants. * p ≤ 0.05; ** p ≤ 0.01. The p values were calculated by one way ANOVA using IgG1 as a control in each concentration group. Shown are the means of three independent samples ± standard deviation. MFI: median fluorescence intensity; a.u.: arbitrary units.</p