Engineering repeat proteins of the immune system

Limitations associated with immunoglobulins have motivated the search for novel binding scaffolds. Repeat proteins have emerged as one promising class of scaffolds, but often are limited to binding protein and peptide targets. An exception is the repeat proteins of the immune system, which have in recent years served as an inspiration for binding scaffolds which can bind glycans and other classes of biomolecule. Like other repeat proteins, these proteins can be very stable and have a monomeric mode of binding, with elongated and highly variable binding surfaces. The ability to target glycans and glycoproteins fill an important gap in current tools for research and biomedical applications.     

B‐cell capacity for differentiation changes with age

BackgroundAge‐related immune deficiencies are thought to be responsible for increased susceptibility to infection in older adults, with alterations in lymphocyte populations becoming more prevalent over time. The loss of humoral immunity in ageing was attributed to the diminished numbers of B cells and the reduced ability to generate immunoglobulin.AimsTo compare the intrinsic B‐cell capacity for differentiation into mature plasma cells (PCs), between young and old donors, using in vitro assays, providing either effective T‐cell help or activation via TLR engagement.MethodsB cells were isolated from healthy individuals, in younger (30–38 years) and older (60–64 years) donors. An in vitro model system of B‐cell differentiation was used, analysing 5 differentiation markers by flow cytometry, under T‐dependent (TD: CD40/BCR stimulation) or T‐independent (TI: TLR7/BCR activation) conditions. Antibody secretion was measured by ELISA and gene expression using qPCR.ResultsTI and TD differentiation resulted in effective proliferation of B cells followed by their differentiation into PC. B‐cell‐executed TI differentiation was faster, all differentiation marker and genes being expressed earlier than under TD differentiation (day 6), although generating less viable cells and lower antibody levels (day 13). Age‐related differences in B‐cell capacity for differentiation were minimal in TD differentiation. In contrast, in TI differentiation age significantly affected proliferation, viability, differentiation, antibody secretion and gene expression, older donors being more efficient.ConclusionAltogether, B‐cell differentiation into PC appeared similar between age groups when provided with T‐cell help, in contrast to TI differentiation, where multiple age‐related changes suggest better capacities in older donors. These new findings may help explain the emergence of autoantibodies in ageing.     

A simple,band‐mounted device to measure behaviorally modified thermal microclimates experienced by birds

Birds encounter climate at the scale of microclimates that can vary rapidly in time and space and so understanding potential vulnerability and adaptations to those microclimates requires fine‐scale measurements that accurately track thermal exposures. However, few options exist for recording the microclimates actually experienced by birds (realized microclimates). We constructed and tested a simple, low‐cost, temperature logger for recording the realized microclimates of ground‐nesting birds. We developed attachment protocols for band‐mounting Thermochron iButtons on Ring‐billed Gull (Larus delawarensis) chicks. We tested these mounted, temperature‐logging devices on 20 chicks weighing > 200 g (device weight was 4 g), attaching devices for 48 h and observing behavior before and after attachment and removal. Devices recorded temperature immediately surrounding the leg at 2‐min intervals. Recorded temperatures were strong predictors of observed thermoregulatory behaviors (panting and sitting), outperforming predictions based on air temperatures measured by basic, static approaches. Through comparison with matched controls (chicks with just a band), we detected no adverse physiological effects of devices, no effects on social or feeding behavior, and only a short‐term decrease in inactivity immediately after device attachment (likely due to increased preening). By attaching iButtons to the legs of birds, we quantified realized thermal exposure, integrating air temperature, modes of environmental heat transfer, and bird behavior at microclimatic scales. Although not yet validated for broader use, our approach (including possible miniaturization) should be suitable to measure thermal exposure of adults, not just chicks, allowing collection of data concerning thermal exposures during flight under field conditions. At ~ $25 USD per device, our approach facilitates experimental protocols with robust sample sizes, even for relatively modest budgets.     

Demonstrating the Potential for Dynamic Auditory Stimulation to Contribute to Motion Sickness

Auditory cues can create the illusion of self-motion (vection) in the absence of visual or physical stimulation. The present study aimed to determine whether auditory cues alone can also elicit motion sickness and how auditory cues contribute to motion sickness when added to visual motion stimuli. Twenty participants were seated in front of a curved projection display and were exposed to a virtual scene that constantly rotated around the participant''s vertical axis. The virtual scene contained either visual-only, auditory-only, or a combination of corresponding visual and auditory cues. All participants performed all three conditions in a counterbalanced order. Participants tilted their heads alternately towards the right or left shoulder in all conditions during stimulus exposure in order to create pseudo-Coriolis effects and to maximize the likelihood for motion sickness. Measurements of motion sickness (onset, severity), vection (latency, strength, duration), and postural steadiness (center of pressure) were recorded. Results showed that adding auditory cues to the visual stimuli did not, on average, affect motion sickness and postural steadiness, but it did reduce vection onset times and increased vection strength compared to pure visual or pure auditory stimulation. Eighteen of the 20 participants reported at least slight motion sickness in the two conditions including visual stimuli. More interestingly, six participants also reported slight motion sickness during pure auditory stimulation and two of the six participants stopped the pure auditory test session due to motion sickness. The present study is the first to demonstrate that motion sickness may be caused by pure auditory stimulation, which we refer to as “auditorily induced motion sickness”.     

Free Edges in Epithelial Cell Sheets Stimulate Epidermal Growth Factor Receptor Signaling

The ability of epithelia to migrate and cover wounds is essential to maintaining their functions as physical barriers. Wounding induces many cues that may affect the transition to motility, including the immediate mechanical perturbation, release of material from broken cells, new interactions with adjacent extracellular matrix, and breakdown of physical separation of ligands from their receptors. Depending on the exact nature of wounds, some cues may be present only transiently or insignificantly. In many epithelia, activation of the epidermal growth factor receptor (EGFR) is a central event in induction of motility, and we find that its continuous activation is required for progression of healing of wounds in sheets of corneal epithelial cells. Here, we examine the hypothesis that edges, which are universally and continuously present in wounds, are a cue. Using a novel culture model we find that their presence is sufficient to cause activation of the EGFR and increased motility of cells in the absence of other cues. Edges that are bordered by agarose do not induce activation of the EGFR, indicating that activation is not due to loss of any specific type of cell–cell interaction but rather due to loss of physical constraints.     

Phosphorylation of the acetylcholine receptor by protein kinase C and identification of the phosphorylation site within the receptor delta subunit

Purified acetylcholine receptor is rapidly and specifically phosphorylated by partially purified protein kinase C, the Ca2+/phospholipid-dependent enzyme. The receptor delta subunit is the major target for phosphorylation and is phosphorylated on serine residues to a final stoichiometry of 0.4 mol of phosphate/mol of subunit. Phosphorylation is dose-dependent with a Km value of 0.2 microM. Proteolytic digestion of the delta subunit phosphorylated by either protein kinase C or the cAMP-dependent protein kinase yielded a similar pattern of phosphorylated fragments. The amino acids phosphorylated by either kinase co-localized within a 15-kDa proteolytic fragment of the delta subunit. This fragment was visualized by immunoblotting with antibodies against a synthetic peptide corresponding to residues 354-367 of the receptor delta subunit. This sequence, which contains 3 consecutive serine residues, was recently shown to include the cAMP-dependent protein kinase phosphorylation site (Souroujon, M. C., Neumann, D., Pizzighella, S., Fridkin, M., and Fuchs, S. (1986) EMBO J. 5, 543-546). Concomitantly, the synthetic peptide 354-367 was specifically phosphorylated in a Ca2+- and phospholipid-dependent manner by protein kinase C. Furthermore, antibodies directed against this peptide inhibited phosphorylation of the intact receptor by protein kinase C. We thus conclude that both the cAMP-dependent protein kinase and protein kinase C phosphorylation sites reside in very close proximity within the 3 adjacent serine residues at positions 360, 361, and 362 of the delta subunit of the acetylcholine receptor.