Comparative Dissociation of Peptide Polyanions by Electron Impact and Photo-Induced Electron Detachment

We compare product-ion mass spectra produced by electron detachment dissociation (EDD) and electron photodetachment dissociation (EPD) of multi-deprotonated peptides on a Fourier transform and a linear ion trap mass spectrometer, respectively. Both methods, EDD and EPD, involve the electron emission-induced formation of a radical oxidized species from a multi-deprotonated precursor peptide. Product-ion mass spectra display mainly fragment ions resulting from backbone cleavages of Cα–C bond ruptures yielding a and x ions. Fragment ions originating from N–Cα backbone bond cleavages are also observed, in particular by EPD. Although EDD and EPD methods involve the generation of a charge-reduced radical anion intermediate by electron emission, the product ion abundance distributions are drastically different. Both processes seem to be triggered by the location and the recombination of radicals (both neutral and cation radicals). Therefore, EPD product ions are predominantly formed near tryptophan and histidine residues, whereas in EDD the negative charge solvation sites on the backbone seem to be the most favorable for the nearby bond dissociation

Mammalian Cell Viability Methods in 3D Scaffolds for Tissue Engineering

Modern methods have evolved in tissue engineering to evaluate cell viability (CV) in 3D scaffolds and tissues. These involve either the usage of 3D confocal laser microscopy of live or fixed tissues, or separation of cells from the tissue, either live or fixed, and then their analysis by flow cytometry. Generally, working with live cells has the disadvantage that all the scanning needs to be completed immediately at the end of an experiment. Two different approaches can be distinguished: staining intact cell membranes and staining fixed cells. The entire cytoplasm is stained with amine-reactive dyes (ARDs), these use the principle of dead cell exclusion. Here, we list and compare live-cell versus fixed-cells fluorescence-based methods and also show their limitations, especially when working with autofluorescent or cross-linking materials like silk or genipin-reinforced hydrogels. Microscopic techniques have the advantage over flow cytometry-based methods in that these provide the spatial distribution and morphology of the cells. Calcein AM combined with ethidium-homodimer works for most 3D constructs, where no strong fluorescent background is found on the tissue or scaffold. Frequently, however, concentrations and incubation times need to be adjusted for a specific tissue to ensure diffusion of dyes and optimise emittance for detection

A Two-pronged Binding Mechanism of IgG to the Neonatal Fc Receptor Controls Complex Stability and IgG Serum Half-life

The success of recombinant monoclonal immunoglobulins (IgG) is rooted in their ability to target distinct antigens with high affinity combined with an extraordinarily long serum half-life, typically around 3 weeks. The pharmacokinetics of IgGs is intimately linked to the recycling mechanism of the neonatal Fc receptor (FcRn). For long serum half-life of therapeutic IgGs, the highly pH-dependent interaction with FcRn needs to be balanced to allow efficient FcRn binding and release at slightly acidic pH and physiological pH, respectively. Some IgGs, like the antibody briakinumab has an unusually short half-life of ∼8 days. Here we dissect the molecular origins of excessive FcRn binding in therapeutic IgGs using a combination of hydrogen/deuterium exchange mass spectrometry and FcRn affinity chromatography. We provide experimental evidence for a two-pronged IgG-FcRn binding mechanism involving direct FcRn interactions with both the Fc region and the Fab regions of briakinumab, and correlate the occurrence of excessive FcRn binding to an unusually strong Fab-FcRn interaction

Structure-Based Prediction of Asparagine and Aspartate Degradation Sites in Antibody Variable Regions

Monoclonal antibodies (mAbs) and proteins containing antibody domains are the most prevalent class of biotherapeutics in diverse indication areas. Today, established techniques such as immunization or phage display allow for an efficient generation of new mAbs. Besides functional properties, the stability of future therapeutic mAbs is a key selection criterion which is essential for the development of a drug candidate into a marketed product. Therapeutic proteins may degrade via asparagine (Asn) deamidation and aspartate (Asp) isomerization, but the factors responsible for such degradation remain poorly understood. We studied the structural properties of a large, uniform dataset of Asn and Asp residues in the variable domains of antibodies. Their structural parameters were correlated with the degradation propensities measured by mass spectrometry. We show that degradation hotspots can be characterized by their conformational flexibility, the size of the C-terminally flanking amino acid residue, and secondary structural parameters. From these results we derive an accurate in silico prediction method for the degradation propensity of both Asn and Asp residues in the complementarity-determining regions (CDRs) of mAbs

Ostertagia spp., rumen fluke and liver fluke single- and poly-infections in cattle: an abattoir study of prevalence and production impacts in England and Wales

This study aims at investigating the occurrence, risk factors and production impacts on beef carcassparameters of three of the most important cattle helminth infections in England and Wales. Abomasa, reticulorumens and livers from healthy cattle were collected and examined post-mortem quarterly over a one year period in an abattoir in South-West England. Specific viscera from 974 cattle were collected,examined and scored for Ostertagia spp., adult rumen fluke and liver fluke lesions/presence. A total of89%, 25% and 29% of the carcasses had lesions/presence of Ostertagia spp., rumen fluke and liver fluke, respectively, and 39% had presence of helminth co-infection. Animal demographic and carcass parameters associated with helminth infections were investigated using multi level multinomial and multilevel linear mixed models respectively. After adjusting for other factors, significant differences in the distribution of helminth infections were observed among cattle by type of breed, animal category (cow, heifer, steer and young bull), age, season and concurrent helminth infections. Compared to carcasses free of helminths, carcasses presenting solely Ostertagia Spp. lesions or adult rumen fluke had significantly lower cold carcass weight (coef.: −30.58 [−50.92;−10.24] and −50.34 [−88.50;−12.18]) and fat cover-age (coef.: −3.28 [−5.56;−1.00] and −5.49 [−10.28;−0.69]) and carcasses presenting solely liver flukelesions had significantly lower conformation grade (coef.: −3.65 [−6.98;−0.32]). Presence of helminth poly-infections was negatively associated with cold carcass weight

Contribution à l'étude de la pathologie du gros intestin. Le petit entéro-colisme (Etude clinique)

Thèse : Médecine : Université de Bordeaux : 1910N° d'ordre : 3

Dimensionnement et enclenchement des bobines d'inductance (selfs) moyenne tension

International audienceThis paper presents a method for the sizing of a gapped-core nonlinear shunt reactor used for mitigation of overvoltages in a medium voltage distribution network with a high level of distributed generation. The study of the transient phenomena that occurs during reactor switching-in is also carried out. These phenomena include (i) inrush currents during switching due to saturation of the magnetic core, which is a silicon-steel (SiFe) iron core, and (ii) also the resulting voltage drop due to these high currents. The study shows the design a 1.4 Mvar shunt reactor with steps of 0.2 Mvar and presents inrush currents for the various steps.Cet article présente une méthode pour le dimensionnement de selfs shunt non-linéaires avec entrefer utilisés pour limiter les surtensions dans un réseau de distribution de moyenne tension avec un niveau élevé de production décentralisée. L'étude des phénomènes transitoires qui se produisent pendant l'enclenchement du réacteur est également réalisée. Ces phénomènes comprennent (i) les courants d'appel pendant la commutation en raison de la saturation du noyau magnétique, qui est un noyau de fer-silicium (SiFe), et (ii) également la chute de tension qui en résulte en raison de ces courants élevés. L'étude montre la conception d'un réacteur shunt de 1,4 Mvar avec des gradins de 0,2 Mvar

Boosting the Liquid Chromatography Separation of Complex Bispecific Antibody Products by Using the Multi-Isocratic Elution Mode

In reversed-phase liquid chromatography (RPLC), the selectivity between major species and minor variants of protein biopharmaceutical products is always limited. Unfortunately, the stationary phase chemistry, type of mobile phase (organic modifier and salts) and temperature only have a very limited impact on selectivity. Therefore, instead of using a linear elution gradient, we evaluated a recently developed strategy, named the multi-isocratic elution mode, to improve the chromatographic resolution. In this contribution, a generic workflow involving the use of an Excel spreadsheet is provided for the rapid and successful development of multi-isocratic elution methods, without the need to use HPLC modeling software. This simple strategy was then successfully applied to very complex biopharmaceutical products; these included one reduced mAb-cytokine fusion protein and a mAb-domain-fusion (C-terminal) protein sample, containing numerous minor variants that were poorly separated from the major species. The addition of several isocratic steps during the chromatographic run provides a clear added value in terms of chromatographic selectivity for several variants, simplifying characterization of the sample with advanced MS tools. In addition to these advantages, some of the limitations of the multi-isocratic elution mode were also highlighted; these included the need to use a highly precise pumping device (preferably, a binary pumping system) and the need to prepare highly accurate mobile phases.</p

Unusual acute neonatal mortality and sow agalactia linked with ergot alkaloid contamination of feed

Background:An increase in the occurrence of ergot alkaloid contamination has been observed in Europe in recentyears. The typical clinical signs of pig ergot poisoning are impaired growth, agalactia and, sometimes, gangrene.Opportunities for reporting exposure doses associated with clinical signs in animals under field conditions are rare.Case presentation:In a farrow-to-finish pig farm with 160 sows, excessive acute neonatal mortality was reportedin association with a loss of appetite and agalactia in sows. A herd examination was conducted and a high rate ofpiglet loss and agalactia in 13 sows out of the most affected batch of 20 were confirmed. Necropsy showed pigletswith empty stomachs and intestines, with apparently normal mucosa. Gestating and lactating sow diet samples, aswell as a wheat sample, were sent for analysis following feed mill inspection and a hypothesis of mycotoxincontamination of self-prepared feed. Liquid chromatography with mass spectrometry in tandem revealed anamount of total ergot alkaloids in all of the samples ranging from 3.49 mg/kg (gestating diet) to 8.06 mg/kg(lactating diet). The contaminated feed was removed and the situation returned to normal 3 weeks later (followingbatch of sows).Conclusion:In the present case, the exposure of sows to 3.49 mg/kg ergot alkaloid for 10 to 15 days before theend of gestation and to 8.06 mg/kg ergot alkaloid over 3 to 4 days at the beginning of lactation - corresponding toa content of 10,146 mg of sclerotia/kg in the wheat of the diets- led to agalactia in 13 of 20 sows in a batch and toa high neonatal mortality rates for all litters. No clinical signs associated with vasoconstrictive effects were observed