Alzheimer’s disease (AD) is a neurodegenerative disorder that causes cognitive decline, memory loss, and inability to perform everyday functions. Hallmark features of AD—including generation of amyloid plaques, neurofibrillary tangles, gliosis, and inflammation in the brain—are well defined; however, the cause of the disease remains elusive. Growing evidence implicates pathogens in AD development, with herpes simplex virus type I (HSV-1) gaining increasing attention as a potential causative agent. Here, we describe a multidisciplinary approach to produce physiologically relevant human tissues to study AD using human-induced neural stem cells (hiNSCs) and HSV-1 infection in a 3D bioengineered brain model. We report a herpes-induced tissue model of AD that mimics human disease with multicellular amyloid plaque–like formations, gliosis, neuroinflammation, and decreased functionality, completely in the absence of any exogenous mediators of AD. This model will allow for future studies to identify potential downstream drug targets for treating this devastating disease.
Repeat proteins have recently emerged as especially well-suited alternative binding scaffolds due to their modular architecture and biophysical properties. Here we present the design of a scaffold based on the consensus sequence of the leucine rich repeat (LRR) domain of the NOD family of cytoplasmic innate immune system receptors. Consensus sequence design has emerged as a protein design tool to create de novo proteins that capture sequence-structure relationships and interactions present in nature. The multiple sequence alignment of 311 individual LRRs, which are the putative ligand-recognition domain in NOD proteins, resulted in a consensus sequence protein containing two internal and N-and C-capping repeats named CLRR2. CLRR2 protein is a stable, monomeric, and cysteine free scaffold that without any affinity maturation displays micromolar binding to muramyl dipeptide, a bacterial cell wall fragment. To our knowledge, this is the first report of direct interaction of a NOD LRR with a physiologically relevant ligand.
Mesoscaled assemblies are organized in native collagen tissues to achieve remarkable and diverse performance and functions. In this work, a facile, low-cost, and controllable liquid exfoliation method was applied to directly extract these collagen mesostructures from bovine Achilles tendons using a sodium hydroxide (NaOH)/urea aqueous system with freeze−thaw cycles and sonication. A series of collagen fibrils with diameters of 26−230 nm were harvested using this process, and in situ observations under polarizing microscopy (POM) and using molecular dynamics simulations revealed the influence of the NaOH/urea system on the tendon collagen. FTIR and XRD results confirmed that these collagen fibrils preserved typical structural characteristics of type I collagen. These isolated collagen fibrils were then utilized as building blocks to fabricate free-standing collagen membranes, which exhibited good stability in solvents and outstanding mechanical properties and transparency, with potential for utility in optical and electronic sensors. Moreover, in vitro and vivo evaluations demonstrated that these new resulting collagen membranes had good cytocompatibility, biocompatibility, and degradability for potential applications in biomedicine. This work provides a new approach for collagen processing by liquid exfoliation with utility for the formation of robust collagen materials that consist of native collagen mesostructures as building blocks.
Three‐dimensional organoid tissue culture models are a promising approach for the study of biological processes including diseases. Advances in these tissue culture technologies improve in vitro analysis compared to standard 2D cellular approaches and are more representative of the physiological environment. However, a major challenge associated with organoid systems stems from the laborious processing involved in the analysis of large numbers of organoids. Here the design, characterization, and application of silk‐elastin‐like protein‐based smart carrier arrays for processing organoids is presented. Fabrication of hydrogel‐based carrier systems at room temperature result in organized arrays of organoids that maintain tissue culture plate orientation and could be processed simultaneously for histology. The system works by transfer of the organoids to the hydrogel arrays after which the material is subjected to 65 °C to induce hydrogel contraction to secure the organoids, resulting in multisample constructs and allowing for placement on a microscope slide. Histological processing and immunostaining of these arrayed cerebral organoids analyzed within the contracted silk‐elastin‐like proteins (SELP) show retention of native organoid features compared to controls without the hydrogel carrier system, thus avoiding any artifacts. These SELP carriers present a useful approach for improving efficiency of scaled organoid screening and processing.
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