Correction: Addition of angled rungs to the horizontal ladder walking task for more sensitive probing of sensorimotor changes.

[This corrects the article DOI: 10.1371/journal.pone.0246298.].

Addition of angled rungs to the horizontal ladder walking task for more sensitive probing of sensorimotor changes.

One method for the evaluation of sensorimotor therapeutic interventions, the horizontal ladder walking task, analyzes locomotor changes that may occur after disease, injury, or by external manipulation. Although this task is well suited for detection of large effects, it may overlook smaller changes. The inability to detect small effect sizes may be due to a neural compensatory mechanism known as "cross limb transfer", or the contribution of the contralateral limb to estimate an injured or perturbed limb's position. The robust transfer of compensation from the contralateral limb may obscure subtle locomotor outcomes that are evoked by clinically relevant therapies, in the early onset of disease, or between higher levels of recovery. Here, we propose angled rungs as a novel modification to the horizontal ladder walking task. Easily-adjustable angled rungs force rats to locomote across a different locomotion path for each hindlimb and may therefore make information from the contralateral limb less useful. Using hM3Dq (excitatory) Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) expressed in large diameter peripheral afferents of the hindlimb in the intact animal, we characterized the sensitivity of our design to detect stepping differences by comparing locomotor changes observed on angled rungs to those observed on a standard horizontal ladder. On our novel asymmetrical ladder, activation of DREADDs resulted in significant differences in rung misses (p = 0.000011) and weight-supporting events (p = 0.049). By comparison, on a standard ladder, we did not observe differences in these parameters (p = 0.86 and p = 0.98, respectively). Additionally, no locomotor differences were detected in baseline and inactivated DREADDs trials when we compared ladder types, suggesting that the angled rungs do not change animal gait behavior unless intervention or injury is introduced. Significant changes observed with angled rungs may demonstrate more sensitive probing of locomotor changes due to the decoupling of cross limb transfer.

Soy Protein Nanofiber Scaffolds for Uniform Maturation of Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium.

Retinal pigment epithelium (RPE) differentiated from human induced pluripotent stem cells, called induced retinal pigment epithelium (iRPE), is being explored as a cell-based therapy for the treatment of retinal degenerative diseases, especially age-related macular degeneration. The success of RPE implantation is linked to the use of biomimetic scaffolds that simulate Bruch's membrane and promote RPE maturation and integration as a functional tissue. Due to difficulties associated with animal protein-derived scaffolds, including sterility and pro-inflammatory responses, current practices favor the use of synthetic polymers, such as polycaprolactone (PCL), for generating nanofibrous scaffolds. In this study, we tested the hypothesis that plant protein-derived fibrous scaffolds can provide favorable conditions permissive for the maturation of RPE tissue sheets in vitro. Our natural, soy protein-derived nanofibrous scaffolds exhibited a J-shaped stress-strain curve that more closely resembled the mechanical properties of native tissues than PCL with significantly higher hydrophilicity of the natural scaffolds, favoring in vivo implantation. We then demonstrate that iRPE sheets growing on these soy protein scaffolds are equivalent to iRPE monolayers cultured on synthetic PCL nanofibrous scaffolds. Immunohistochemistry demonstrated RPE-like morphology and functionality with appropriate localization of RPE markers RPE65, PMEL17, Ezrin, and ZO1 and with anticipated histotypic polarization of vascular endothelial growth factor and pigment epithelium-derived growth factor as indicated by enzyme-linked immunosorbent assay. Scanning electron microscopy revealed dense microvilli on the cell surface and homogeneous tight junctional contacts between the cells. Finally, comparative transcriptome analysis in conjunction with principal component analysis demonstrated that iRPE on nanofibrous scaffolds, either natural or synthetic, matured more consistently than on nonfibrous substrates. Taken together, our studies suggest that the maturation of cultured iRPE sheets for subsequent clinical applications might benefit from the use of nanofibrous scaffolds generated from natural proteins. Impact statement Induced retinal pigment epithelium (iRPE) from patient-derived induced pluripotent stem cells (iPSCs) may yield powerful treatments of retinal diseases, including age-related macular degeneration. Recent studies, including early human clinical trials, demonstrate the importance of selecting appropriate biomaterial scaffolds to support tissue-engineered iRPE sheets during implantation. Electrospun scaffolds show particular promise due to their similarity to the structure of the native Bruch's membrane. In this study, we describe the use of electroprocessed nanofibrous soy protein scaffolds to generate polarized sheets of human iPSC-derived iRPE sheets. Our evaluation, including RNA-seq transcriptomics, indicates that these scaffolds are viable alternatives to scaffolds electrospun from synthetic polymers.

An Air Bubble-Isolating Rotating Wall Vessel Bioreactor for Improved Spheroid/Organoid Formation.

IMPACT STATEMENT: The rotating wall vessel (RWV) bioreactor is a powerful tool for the generation of sizeable, faster-growing organoids. However, the ideal, low-shear, modeled microgravity environment in the RWV is frequently disrupted by the formation of bubbles, a critical but understated failure mode. To address this, we have designed and fabricated a novel, modified RWV bioreactor capable of continuously removing bubbles while providing optimal fluid dynamics. We validated the capacity of this device with computational and empirical studies. We anticipate that our novel bioreactor will be more consistent and easier to use and may fill a unique and unmet niche in the burgeoning field of organoids.