On-line monitoring and controlling of cell apoptosis in mammalian cell culture processes using dielectric spectroscopy

We investigate a method to control critical quality attributes and apply Process Analytical Technology (PAT) via online dielectric spectroscopy (DS) feedback. This system has been intensively explored and successfully implemented in GMP manufacturing processes at Biogen. The present bioreactor application however, is basic and only allows the prediction of biomass. To further enhance the cell culture process robustness, we investigated the feasibility of using the full-spectrum dielectric spectroscopy scanning function to detect dielectric property changes in the cells associated with shifts in cell health and/or metabolism. In this proof of concept study, we used several CHO cell processes to demonstrate that DS probes can be used to not only measure the biomass but also reflect the cell’s physiological state changes (e.g. cell apoptosis). The results showed that one or more of the key parameters of delta capacitance (De), critical frequency (fc), and Cole-Cole Alpha (a) from the multi-frequency scanning data could reflect the cell’s early apoptosis induced by chemical treatment, nutrient depletion, or shear stress, which were seen earlier than that obtained from off-line methods (e.g. trypan blue exclusion). In some cases, by responding to the earlier detection, the cell apoptosis was reversed in time and the batch was saved. This enables a potential application, transferrable across programs, of full-spectrum dielectric spectroscopy for earlier detection of physiological changes, allowing for timelier bioreactor process adjustments. In addition, the feasibility of the application of multifrequency scanning in cGMP process for monitoring and control was also explored in this study

Designing libraries of chimeric proteins using SCHEMA recombination and RASPP

SCHEMA is a method for designing libraries of novel proteins by recombination of homologous sequences. The goal is to maximize the number of folded proteins while simultaneously generating significant sequence diversity. Here, we use the RASPP algorithm to identify optimal SCHEMA designs for shuffling contiguous elements of sequence. To exemplify the method, SCHEMA is used to recombine five fungal cellobiohydrolases (CBH1s) to produce a library of more than 390,000 novel CBH1 sequences

Amyloid-β nanotubes are associated with prion protein-dependent synaptotoxicity

Growing evidence suggests water-soluble, non-fibrillar forms of amyloid-β protein (Aβ) have important roles in Alzheimer's disease with toxicities mimicked by synthetic Aβ1-42. However, no defined toxic structures acting via specific receptors have been identified and roles of proposed receptors, such as prion protein (PrP), remain controversial. Here we quantify binding to PrP of Aβ1-42 after different durations of aggregation. We show PrP-binding and PrP-dependent inhibition of long-term potentiation (LTP) correlate with the presence of protofibrils. Globular oligomers bind less avidly to PrP and do not inhibit LTP, whereas fibrils inhibit LTP in a PrP-independent manner. That only certain transient Aβ assemblies cause PrP-dependent toxicity explains conflicting reports regarding the involvement of PrP in Aβ-induced impairments. We show that these protofibrils contain a defined nanotubular structure with a previously unidentified triple helical conformation. Blocking the formation of Aβ nanotubes or their interaction with PrP might have a role in treatment of Alzheimer's disease

Investigation of exchange coupled bilayer Fe/CuMnAs by pump-probe experiment

Time-resolved pump-probe magneto-optical method was used to study Fe/CuMnAs bilayer. The probe polarization dependence was used to identify and separate parts of detected signals due to Faraday and Voigt magneto-optical effects that provide information about pump-induced magnetization precession in ferromagnetic (FM) Fe and demagnetization in antiferromagnetic (AF) CuMnAs layers, respectively. We observed a 180 deg phase shift in the precession signal at non-zero magnetic field that we interpreted as a signature of the magnetic anisotropy induced in the FM by the adjacent AF layer. Unlike in x-ray magnetic linear dichroism experiments, we did not observe any significant reorientation of magnetic moments in CuMnAs by external magnetic field due to the interlayer exchange coupling with Fe. Differences between these two experimental techniques, providing the distinct pictures, are discussed

Baby-Led Weaning: The Evidence to Date

Purpose of ReviewInfants are traditionally introduced to solid foods using spoon-feeding of specially prepared infant foods.Recent FindingsHowever, over the last 10–15 years, an alternative approach termed ‘baby-led weaning’ has grown in popularity. This approach involves allowing infants to self-feed family foods, encouraging the infant to set the pace and intake of the meal. Proponents of the approach believe it promotes healthy eating behaviour and weight gain trajectories, and evidence is starting to build surrounding the method. This review brings together all empirical evidence to date examining behaviours associated with the approach, its outcomes and confounding factors.SummaryOverall, although there is limited evidence suggesting that a baby-led approach may encourage positive outcomes, limitations of the data leave these conclusions weak. Further research is needed, particularly to explore pathways to impact and understand the approach in different contexts and populations

OxyCAP UK: Oxyfuel Combustion - academic Programme for the UK

The OxyCAP-UK (Oxyfuel Combustion - Academic Programme for the UK) programme was a £2 M collaboration involving researchers from seven UK universities, supported by E.On and the Engineering and Physical Sciences Research Council. The programme, which ran from November 2009 to July 2014, has successfully completed a broad range of activities related to development of oxyfuel power plants. This paper provides an overview of key findings arising from the programme. It covers development of UK research pilot test facilities for oxyfuel applications; 2-D and 3-D flame imaging systems for monitoring, analysis and diagnostics; fuel characterisation of biomass and coal for oxyfuel combustion applications; ash transformation/deposition in oxyfuel combustion systems; materials and corrosion in oxyfuel combustion systems; and development of advanced simulation based on CFD modelling

Identification of a Compound That Disrupts Binding of Amyloid-β to the Prion Protein Using a Novel Fluorescence-based Assay

The prion protein (PrP) has been implicated both in prion diseases such as Creutzfeldt-Jakob disease, where its monomeric cellular isoform (PrP(C)) is recruited into pathogenic self-propagating polymers of misfolded protein, and in Alzheimer disease, where PrP(C) may act as a receptor for synaptotoxic oligomeric forms of amyloid-β (Aβ). There has been considerable interest in identification of compounds that bind to PrP(C), stabilizing its native fold and thereby acting as pharmacological chaperones to block prion propagation and pathogenesis. However, compounds binding PrP(C) could also inhibit the binding of toxic Aβ species and may have a role in treating Alzheimer disease, a highly prevalent dementia for which there are currently no disease-modifying treatments. However, the absence of a unitary, readily measurable, physiological function of PrP makes screening for ligands challenging, and the highly heterogeneous nature of Aβ oligomer preparations makes conventional competition binding assays difficult to interpret. We have therefore developed a high-throughput screen that utilizes site-specifically fluorescently labeled protein to identify compounds that bind to PrP and inhibit both Aβ binding and prion propagation. Following a screen of 1,200 approved drugs, we identified Chicago Sky Blue 6B as the first small molecule PrP ligand capable of inhibiting Aβ binding, demonstrating the feasibility of development of drugs to block this interaction. The interaction of Chicago Sky Blue 6B was characterized by isothermal titration calorimetry, and its ability to inhibit Aβ binding and reduce prion levels was established in cell-based assays

Exploiting inflammation for therapeutic gain in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy associated with <5% 5-year survival, in which standard chemotherapeutics have limited benefit. The disease is associated with significant intra- and peritumoral inflammation and failure of protective immunosurveillance. Indeed, inflammatory signals are implicated in both tumour initiation and tumour progression. The major pathways regulating PDAC-associated inflammation are now being explored. Activation of leukocytes, and upregulation of cytokine and chemokine signalling pathways, both have been shown to modulate PDAC progression. Therefore, targeting inflammatory pathways may be of benefit as part of a multi-target approach to PDAC therapy. This review explores the pathways known to modulate inflammation at different stages of tumour development, drawing conclusions on their potential as therapeutic targets in PDAC

Characterising pulverised fuel ignition in a visual drop tube furnace by use of a high-speed imaging technique

This study investigates the ignition characteristics of pulverised coal, biomass and co-firing by use of a visual drop tube furnace (VDTF) and a high speed imaging technique. Three coals (anthracite, a bituminous coal and a lignite), four biomasses (Pine, Eucalyptus, Olive Residue and Miscanthus) and various biomass-coal mixtures were tested. With each coal, biomass or their mixture, a distinct flame was established within the VDTF through the continuous feeding of the fuel under the environment of air and at a furnace temperature of 800 °C. To observe the ignition point, a Phantom v12.1 high-speed camera was used to capture the videos of fuel combustion at 500 frames per second (FPS). A technique was developed using MATLAB's image analysis tool to automate the ignition point detection. The results of the image processing were used to statistically analyse and determine the changes to the ignition behaviour with different fuels and co-firing ratios. The results obtained with the tested coals have shown that the distance to ignition increases as the coal volatile matter content decreases, whereas the opposite trend was found for the biomass fuels. Further, the addition of biomass to the anthracite significantly reduces the distance to ignition but a much less pronounced effect on the ignition was found when biomass was co-fired with the bituminous coal or lignite. The synergistic effect on the ignition of biomass-anthracite mixture is mainly attributed to the high volatile content and the potential effects of catalysis from the alkali metals present in the biomass. The results of this study have shown that the VDTF testing coupled with the image analysis technique allows for an effective and simple method of characterising ignition behaviours of pulverised coal, biomass and their mixtures

Use of the lambda Red recombinase system to rapidly generate mutants in Pseudomonas aeruginosa

<p>Abstract</p> <p>Background</p> <p>The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in various bacteria and fungi. The procedure consists of electroporating a polymerase chain reaction (PCR) fragment that was obtained with a 1- or 3-step PCR protocol and that carries an antibiotic cassette flanked by a region homologous to the target locus into a strain that expresses the lambda Red recombination system.</p> <p>Results</p> <p>This system has been modified for use in <it>Pseudomonas aeruginosa</it>. Chromosomal DNA deletions of single genes were generated using 3-step PCR products containing flanking regions 400–600 nucleotides (nt) in length that are homologous to the target sequence. A 1-step PCR product with a homologous extension flanking region of only 100 nt was in some cases sufficient to obtain the desired mutant. We further showed that the <it>P. aeruginosa </it>strain PA14 non-redundant transposon library can be used in conjunction with the lambda Red technique to rapidly generate large chromosomal deletions or transfer mutated genes into various PA14 isogenic mutants to create multi-locus knockout mutants.</p> <p>Conclusion</p> <p>The lambda Red-based technique can be used efficiently to generate mutants in <it>P. aeruginosa</it>. The main advantage of this procedure is its rapidity as mutants can be easily obtained in less than a week if the 3-step PCR procedure is used, or in less than three days if the mutation needs to be transferred from one strain to another.</p