A Simple Method for the Purification of Nisin

Nisin, an antimicrobial peptide showing activity against a broad range of Gram-positive bacteria, is widely used as a food preservative and has potential as a therapeutic for a range of infectious diseases. Here, we present a simple purification method, based on a salting-out approach, which can produce a powder containing ～33% nisin, from a nisin-producing culture in a whey permeate-based medium. This process removes over 99% of the lactic acid, NaCl, lactose and non-nisin proteins from the cell-free culture supernatant. The approach can also enrich a commonly used commercial nisin preparation over 30-fold to a purity of ～58%. These are higher purities than comparable published methods. The simplicity of this approach facilitates its use in research and also its scale-up.     

Origine et évolution des saurischiens

We propose here a short synthesis of the saurischian evolutionary history. Our knowledge of the diversity and evolution of the saurischian non-avian dinosaurs has increased during the past decade. The generalized use of cladistics has led to various phylogenetic hypotheses, some of them in agreement on the evolution of saurischians, even if some controversy remains. The saurischian evolution is closely linked to two of the five great mass extinctions, which punctuated life history, but probably also to a third, less important, extinction event at the end of the Early Jurassic.     

Different Epidermal Growth Factor Receptor (EGFR) Agonists Produce Unique Signatures for the Recruitment of Downstream Signaling Proteins

The EGF receptor can bind seven different agonist ligands. Although each agonist appears to stimulate the same suite of downstream signaling proteins, different agonists are capable of inducing distinct responses in the same cell. To determine the basis for these differences, we used luciferase fragment complementation imaging to monitor the recruitment of Cbl, CrkL, Gab1, Grb2, PI3K, p52 Shc, p66 Shc, and Shp2 to the EGF receptor when stimulated by the seven EGF receptor ligands. Recruitment of all eight proteins was rapid, dose-dependent, and inhibited by erlotinib and lapatinib, although to differing extents. Comparison of the time course of recruitment of the eight proteins in response to a fixed concentration of each growth factor revealed differences among the growth factors that could contribute to their differing biological effects. Principal component analysis of the resulting data set confirmed that the recruitment of these proteins differed between agonists and also between different doses of the same agonist. Ensemble clustering of the overall response to the different growth factors suggests that these EGF receptor ligands fall into two major groups as follows: (i) EGF, amphiregulin, and EPR; and (ii) betacellulin, TGFα, and epigen. Heparin-binding EGF is distantly related to both clusters. Our data identify differences in network utilization by different EGF receptor agonists and highlight the need to characterize network interactions under conditions other than high dose EGF.     

Taphonomic study of a pleurosternid turtle shell from the Early Cretaceous of Angeac‐Charente,southwest France

The feeding behaviour of crocodilians leads to the formation of particular assemblages by accumulation of carcasses on the bottom or at the edge of water points, in addition to the characteristic marks it leaves on bones, making crocodilians important taphonomic agents. We report here the case of a Pleurosternidae turtle shell, Pleurosternon bullockii (Owen 1842 ), discovered in 2014 in Angeac‐Charente in south‐western France. The shell shows a range of tooth marks left by a crocodyliform. Several elements designate Goniopholis as the most likely author of the marks and support the use of the ‘nutcracker’ technique. Some marks appear to have healed, while others suggest that this P. bullockii individual suffered from shell diseases. Many turtle remains and some ornithomimosaur dinosaur bones from Angeac‐Charente bear marks of a crocodyliform attack.     

Raman based chemometric model development for glycation and glycosylation real time monitoring in a manufacturing scale CHO cell bioreactor process

The Quality by Design (QbD) approach to the production of therapeutic monoclonal antibodies (mAbs) emphasizes an understanding of the production process ensuring product quality is maintained throughout. Current methods for measuring critical quality attributes (CQAs) such as glycation and glycosylation are time and resource intensive, often, only tested offline once per batch process. Process analytical technology (PAT) tools such as Raman spectroscopy combined with chemometric modeling can provide real time measurements process variables and are aligned with the QbD approach. This study utilizes these tools to build partial least squares (PLS) regression models to provide real time monitoring of glycation and glycosylation profiles. In total, seven cell line specific chemometric PLS models; % mono-glycated, % non-glycated, % G0F-GlcNac, % G0, % G0F, % G1F, and % G2F were considered. PLS models were initially developed using small scale data to verify the capability of Raman to measure these CQAs effectively. Accurate PLS model predictions were observed at small scale (5 L). At manufacturing scale (2000 L) some glycosylation models showed higher error, indicating that scale may be a key consideration in glycosylation profile PLS model development. Model robustness was then considered by supplementing models with a single batch of manufacturing scale data. This data addition had a significant impact on the predictive capability of each model, with an improvement of 77.5% in the case of the G2F. The finalized models show the capability of Raman as a PAT tool to deliver real time monitoring of glycation and glycosylation profiles at manufacturing scale.     

Regulation of photosensation by hydrogen peroxide and antioxidants in C. elegans

The eyeless C. elegans exhibits robust phototaxis behavior in response to short-wavelength light, particularly UV light. C. elegans senses light through LITE-1, a unique photoreceptor protein that belongs to the invertebrate taste receptor family. However, it remains unclear how LITE-1 is regulated. Here, we performed a forward genetic screen for genes that when mutated suppress LITE-1 function. One group of lite-1 suppressors are the genes required for producing the two primary antioxidants thioredoxin and glutathione, suggesting that oxidization of LITE-1 inhibits its function. Indeed, the oxidant hydrogen peroxide (H₂O₂) suppresses phototaxis behavior and inhibits the photoresponse in photoreceptor neurons, whereas other sensory behaviors are relatively less vulnerable to H₂O₂. Conversely, antioxidants can rescue the phenotype of lite-1 suppressor mutants and promote the photoresponse. As UV light illumination generates H₂O₂, we propose that upon light activation of LITE-1, light-produced H₂O₂ then deactivates LITE-1 to terminate the photoresponse, while antioxidants may promote LITE-1’s recovery from its inactive state. Our studies provide a potential mechanism by which H₂O₂ and antioxidants act synergistically to regulate photosensation in C. elegans.     

Isolation and characterization of a monoclonal antibody containing an extra heavy-light chain Fab arm

Isolation and characterization of monoclonal antibody (mAb) variants to understand the impact of their structure on function is a typical activity during early-stage candidate selection that contributes to derisking clinical development. In particular, efforts are devoted to characterizing oligomeric variants, owing to their potential immunogenic nature. We report here a mAb variant consisting of a canonical mAb monomer associated in a non-covalent fashion with an antigen-binding fragment (Fab) arm amputated from its Fc domain. The truncated heavy chain is encoded in the cell line genome and is the likely product of a genomic recombination during cell line generation. The addition of the Fab arm results in severe loss of potency, indicating its interaction with the Fab domain of the monomer. The presence of such a variant can easily be mitigated by an adequate purification step.