Particle lithography has been extensively used as a robust and cost-effective method to produce large-area, close-packed arrays of nanometer scale features. Many technological applications, including biosensing, require instead non-close-packed patterns in order to avoid cross-talk between the features. We present a simple, scalable, single-step particle lithography process that employs colloidal self-assembly at liquid-liquid interfaces (SALI) to fabricate regular, open particle lithography masks, where the size of the features (40 to 500 nm) and their separation can be independently controlled between 3 and 10 particle diameters. Finally we show how the process can be practically employed to produce diverse biosensing structures.

No abstract

Background: Single domain variable regions of shark antibodies (V-NARs) are promising biotherapeutic candidates. Results: A V-NAR specific for human serum albumin was humanized, and its crystal structure in complex with the antigen was solved, revealing an unusual recognition mode. Conclusion: Humanization preserved antigen binding properties and activity of the parental shark antibody. Significance: A structural framework for humanization of shark antibodies was established.

The transforming growth factor ␤ family member activin is an important regulator of development and tissue repair. It is strongly up-regulated after acute injury to the adult brain, and application of exogenous activin protects neurons in several lesion models. To explore the role of endogenous activin in the normal and acutely damaged brain, we generated transgenic mice expressing a dominant-negative activin receptor IB (dnActRIB) mutant in forebrain neurons. The functionality of the transgene was verified in vivo. Hippocampal neurons from dnActRIB mice were significantly more vulnerable to intracerebroventricular injection of the excitotoxin kainic acid than those from control littermates, indicating a crucial role of endogenous activin in the rescue of neurons from excitotoxic insult. Because dnActRIB is only expressed in neurons, but not in glial cells, activin affords protection at least in part through a direct action on endangered neurons. Unexpectedly, the transgenic mice also revealed a prominent novel role of activin in glutamatergic neurotransmission in the intact adult brain. Electrophysiologic examination of excitatory synapses onto CA1 pyramidal cells in hippocampal slices of dnActRIB mice showed a reduced NMDA current response, which was associated with impaired long term potentiation. This is the first demonstration that activin receptor signaling is essential to optimize the performance of neuronal circuits in the mature brain under physiological conditions. Activins are members of the transforming growth factor ␤ family of proteins, which regulate proliferation and differentiation of various cell types. The predominant activin variants are homo-or heterodimers consisting of ␤A and/or ␤B subunits (activin A: ␤A␤A, activin B: ␤B␤B and activin AB: ␤A␤B). Their biological effects are mediated through heteromeric receptor complexes, consisting of type I and type II transmembrane serine/threonine kinase receptors (1). Upon ligand binding, one of the activin type II receptors (ActRII or ActRIIB) dimerizes with a type I receptor (ActRIA, ActRIB, or activin receptor-like kinase 7 (2, 3)), resulting in phosphorylation of the type I receptor by the type II receptor. The subsequently activated serine/ threonine kinase of the type I receptor phosphorylates the recruited receptor Smads (Smad2 and Smad3). The latter translocate to the nucleus upon multimerization with Smad4 and modulate as transcription factor complexes the expression of activin target genes (4). In addition, other signaling pathways are also activated by activin receptors, including mitogen activated kinase signaling (1,4,5).Activins are important regulators of development, inflammation, and repair of different tissues and organs (1, 5). In the central nervous system, activin is involved in development, but it may also participate in adaptive and protective mechanisms of the adult brain. Thus, activin ␤A mRNA is transiently upregulated after electrical stimuli that induce synaptic long term potentiation (LTP 4 ; Refs. 6 and 7) and after brie...

The transforming growth factor-beta family member activin is a potent regulator of skin morphogenesis and repair. Transgenic mice overexpressing activin in keratinocytes display epidermal hyper-thickening and dermal fibrosis in normal skin and enhanced granulation tissue formation after wounding. Mice overexpressing the secreted activin antagonist follistatin, however, have the opposite wound-healing phenotype. To determine whether activin affects skin morphogenesis and repair via activation of keratinocytes and/or stromal cells, we generated transgenic mice expressing a dominant-negative activin receptor IB mutant (dnActRIB) in keratinocytes. The architecture of adult skin was unaltered in these mice, but delays were observed in postnatal pelage hair follicle morphogenesis and in the first catagen-telogen transformation of hair follicles. Although dnActRIB-transgenic mice showed slightly delayed wound re-epithelialization after skin injury, the strong inhibition of granulation tissue formation seen in follistatin-transgenic mice was not observed. Therefore, although endogenous activin appeared to affect skin morphogenesis and repair predominantly via stromal cells, overexpressed activin strongly affected the epidermis. The epidermal phenotype of activin-overexpressing mice was partially rescued by breeding these animals with dnActRIB-transgenic mice. These results demonstrate that activin affects both stromal cells and keratinocytes in normal and wounded skin and that the effect on keratinocytes is dose-dependent in vivo.

Osteoarthritis leads to an alteration in the composition of the synovial fluid, which is associated with an increase in friction and the progressive and irreversible destruction of the articular cartilage. In order to tackle this degenerative disease, there has been a growing interest in the medical field to establish effective, long-term treatments to restore cartilage lubrication after damage. Here we develop a series of graft-copolymers capable of assembling selectively on the degraded cartilage, resurfacing it, and restoring the lubricating properties of the native tissue. These comprise a polyglutamic acid backbone (PGA) coupled to brush-forming, poly-2-methyl-2-oxazoline (PMOXA) side chains, which provide biopassivity and lubricity to the surface, and to aldehyde-bearing tissue-reactive groups, for the anchoring on the degenerated cartilage via Schiff bases. Optimization of the graft-copolymer architecture (i.e., density and length of side chains and amount of tissue-reactive functions) allowed a uniform passivation of the degraded cartilage surface. Graft-copolymer-treated cartilage showed very low coefficients of friction within synovial fluid, reestablishing and in some cases improving the lubricating properties of the natural cartilage. Due to these distinctive properties and their high biocompatibility and stability under physiological conditions, cartilage-reactive graft-copolymers emerge as promising injectable formulations to slow down the progression of cartilage degradation, which characterizes the early stages of osteoarthritis.

Molecular engineering to increase the percentage identity to common human immunoglobulin sequences of non-human therapeutic antibodies and scaffolds has become standard practice. This strategy is often used to reduce undesirable immunogenic responses, accelerating the clinical development of candidate domains. The first humanized shark variable domain (VNAR) was reported by Kovalenko and colleagues and used the anti-human serum albumin (HSA) domain, clone E06, as a model to construct a number of humanized versions including huE06v1.10. This study extends this work by using huE06v1.10 as a template to isolate domains with improved biophysical properties and reduced antigenicity. Random mutagenesis was conducted on huE06v1.10 followed by refinement of clones through an off-rate ranking-based selection on target antigen. Many of these next-generation binders retained high affinity for target, together with good species cross-reactivity. Lead domains were assessed for any tendency to dimerize, tolerance to N- and C-terminal fusions, affinity, stability, and relative antigenicity in human dendritic cell assays. Functionality of candidate clones was verified in vivo through the extension of serum half-life in a typical drug format. From these analyses the domain, BA11, exhibited negligible antigenicity, high stability and high affinity for mouse, rat, and HSA. When these attributes were combined with demonstrable functionality in a rat model of PK, the BA11 clone was established as our clinical candidate.

No abstract