Novel Selection Approaches to Identify Antibodies Targeting Neoepitopes on the C5b6 Intermediate Complex to Inhibit Membrane Attack Complex Formation

The terminal pathway of complement is implicated in the pathology of multiple diseases and its inhibition is, therefore, an attractive therapeutic proposition. The practicalities of inhibiting this pathway, however, are challenging, as highlighted by the very few molecules in the clinic. The proteins are highly abundant, and assembly is mediated by high-affinity protein–protein interactions. One strategy is to target neoepitopes that are present transiently and only exist on active or intermediate complexes but not on the abundant native proteins. Here, we describe an antibody discovery campaign that generated neoepitope-specific mAbs against the C5b6 complex, a stable intermediate complex in terminal complement complex assembly. We used a highly diverse yeast-based antibody library of fully human IgGs to screen against soluble C5b6 antigen and successfully identified C5b6 neoepitope-specific antibodies. These antibodies were diverse, showed good binding to C5b6, and inhibited membrane attack complex (MAC) formation in a solution-based assay. However, when tested in a more physiologically relevant membrane-based assay these antibodies failed to inhibit MAC formation. Our data highlight the feasibility of identifying neoepitope binding mAbs, but also the technical challenges associated with the identification of functionally relevant, neoepitope-specific inhibitors of the terminal pathway.

Roaming pathways and survival probability in real-time collisional dynamics of cold and controlled bialkali molecules

Perfectly controlled molecules are at the forefront of the quest to explore chemical reactivity at ultra low temperatures. Here, we investigate for the first time the formation of the long-lived intermediates in the time-dependent scattering of cold bialkali $$^{23}\hbox {Na}^{87}$$ 23 Na 87 Rb molecules with and without the presence of infrared trapping light. During the nearly 50 nanoseconds mean collision time of the intermediate complex, we observe unconventional roaming when for a few tens of picoseconds either NaRb or $$\hbox {Na}_2$$ Na 2 and $$\hbox {Rb}_2$$ Rb 2 molecules with large relative separation are formed before returning to the four-atom complex. We also determine the likelihood of molecular loss when the trapping laser is present during the collision. We find that at a wavelength of 1064 nm the $$\hbox {Na}_2\hbox {Rb}_2$$ Na 2 Rb 2 complex is quickly destroyed and thus that the $$^{23}\hbox {Na}^{87}$$ 23 Na 87 Rb molecules are rapidly lost.

Divalent ligand-monovalent molecule binding

Simultaneous binding of divalent ligands to two identical molecules is a widespread phenomenon in biology and chemistry. Here, we describe this binding event as a divalent ligand AA that can bind to two identical monovalent molecules B to form the complex AA · B2. Cases where the total concentration [AA]T is either much larger or much smaller than the total concentration [B]T have been studied earlier, but a description of intermediate concentrations is missing. In this paper, we describe the general case of any ratio of ξ ≡ [B]T /[AA]T. We show that the concentration of the intermediate complex AA · B is governed by a cubic equation and discuss several scenarios in which this cubic equation simplifies. Our numerical results, which cover the entire range of 0 < ξ < ∞, are relevant to processes wherein the concentrations of free ligands and proteins both decrease upon binding. Such ligand and protein depletion is expected to be important in cellular contexts, e.g., in antigen detection and in coincidence detection of proteins or lipids.

Effects of desiccation of seeds in nine species with morphophysiological dormancy on germination and embryo growth

Aims In species with morphophysiological seed dormancy (MPD), little is known about the effects of desiccation of imbibed seeds on embryo growth and germination. We studied seed responses to dehydration in nine species with different levels of MPD. Methods For each species, a control test was conducted by keeping seeds permanently hydrated and exposed to the optimal stratification-incubation sequence to promote embryo growth. Simultaneously, tests were run in which seed stratification was interrupted for 1 month by desiccation at room temperature. Important Findings In Clematis vitalba and Ribes alpinum, with nondeep simple MPD, desiccation affected neither embryo growth nor seed viability, but the desiccation led to a decrease of germinative ability in R. alpinum by 16%. The seeds of Narcissus pseudonarcissus subsp. munozii-garmendiae, with deep simple epicotyl MPD, tolerated desiccation in different embryo growth stages, but their germinative ability decreased slightly. The response of species with complex levels of MPD to desiccation was more variable: Delphinium fissum subsp. sordidum, with intermediate complex MPD, and Anthriscus sylvestris and Meum athamanticum, both with deep complex MPD, tolerated desiccation. In contrast, Ribes uva-crispa with nondeep complex MPD, Lonicera pyrenaica with intermediate complex MPD, and Chaerophyllum aureum with deep complex MPD, had diminished germination ability by desiccation. Although seeds of the species with simple levels of MPD tolerated desiccation, those of some species with complex levels were also highly tolerant. Thus, desiccation did not induce secondary dormancy in late embryo growth stages. The desiccation tolerance of imbibed seeds of most of the nine species may show their adaptability to climate change in the Mediterranean region.