Texturization of pea protein isolate by micro compounding.

Twin-screw micro compounding is introduced as a novel technique to process and characterize small plant protein samples under conditions that are relevant for meat analogue processing. Small samples of pea protein isolate (PPI) (5 cm3, corresponding to ∼7 g of hydrated sample) are batch-processed at water contents between 40 and 70 % w/w and temperatures between 90 and 120 °C. Screw speed (100-400 rpm) and residence time (1-9 min) are varied resulting in values of the specific mechanical energy (SME) between ∼20 and 2000 kJ/kg, which is the range relevant for plant protein extrusion. Micro compounding process data provides information on several aspects of the rheological behavior of PPI. Shear thinning behavior is observed for PPI. The viscosity of the PPI during micro compounding was found to exponentially decrease with water content. The temperature dependence is consistent with an Arrhenius-type model. The extruded strands (length: ∼15 cm; diameter: 3.0 ± 0.2 mm) are characterized by scanning electron microscopy (SEM), differential solubility, water holding capacity (WHC), and texture profile analysis (TPA). The hardness as determined from TPA increases linearly with screw speed and residence time, jumps to higher values above the denaturation temperature of the PPI and decreases exponentially with the water content during processing. Micro compounding is found to be a useful technique to convert small plant protein samples at water contents between about 40 and 60 % w/w into texturized matrices and investigate the rheological behavior of plant protein isolates under conditions that are relevant for extrusion processing.

Analysis of residual disease in periocular basal cell carcinoma following hedgehog pathway inhibition: Follow up to the VISORB trial.

Basal cell carcinoma (BCC) is a common skin cancer caused by deregulated hedgehog signaling. BCC is often curable surgically; however, for orbital and periocular BCCs (opBCC), surgical excision may put visual function at risk. Our recent clinical trial highlighted the utility of vismodegib for preserving visual organs in opBCC patients: 67% of patients displayed a complete response histologically. However, further analysis of excision samples uncovered keratin positive, hedgehog active (Gli1 positive), proliferative micro-tumors. Sequencing of pre-treatment tumors revealed resistance conferring mutations present at low frequency. In addition, one patient with a low-frequency SMO W535L mutation recurred two years post study despite no clinical evidence of residual disease. Sequencing of this recurrent tumor revealed an enrichment for the SMO W535L mutation, revealing that vismodegib treatment enriched for resistant cells undetectable by traditional histology. In the age of targeted therapies, linking molecular genetic analysis to prospective clinical trials may be necessary to provide mechanistic understanding of clinical outcomes. Trial Registration: ClinicalTrials.gov Identifier: NCT02436408.

Retinoic Acid Potentiates Orbital Tissues for Inflammation Through NF-κB and MCP-1.

Purpose: The orbit displays unique vulnerability to inflammatory conditions. The most prevalent of these conditions, thyroid eye disease (TED), occurs in up to 50% of patients with Graves' disease (GD). Whereas the pathology of both TED and GD is driven by autoantibodies, it is unclear why symptoms manifest specifically in the orbit. Methods: We performed retinoic acid treatment on both normal and TED patient-derived orbital fibroblasts (OFs) followed by mRNA and protein isolation, quantitative real-time polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay, RNA sequencing, and Western blot analyses. Results: Both normal and TED patient-derived OFs display robust induction of monocyte chemoattractant protein 1 (MCP-1) upon retinoid treatment; TED OFs secrete significantly more MCP-1 than normal OFs. In addition, pretreatment of OFs with thiophenecarboxamide (TPCA-1) inhibits retinoid-induced MCP-1 induction, suggesting an NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells)-dependent mechanism. We also found that treatment with cholecalciferol (vitamin D3) mitigates MCP-1 induction, likely because of competition between retinoic acid receptors (RARs) and vitamin D receptors (VDR) for their common binding partner retinoid nuclear receptors (RXRs). Conclusions: Retinoids that naturally accumulate in orbital adipose tissue can act on orbital fibroblasts to induce the expression of inflammation-associated genes. These data suggest a potential role for retinoids in sensitizing the orbit to inflammation.

Twist3 is required for dedifferentiation during extraocular muscle regeneration in adult zebrafish.

Severely damaged adult zebrafish extraocular muscles (EOMs) regenerate through dedifferentiation of residual myocytes involving a muscle-to-mesenchyme transition. Members of the Twist family of basic helix-loop-helix transcription factors (TFs) are key regulators of the epithelial-mesenchymal transition (EMT) and are also involved in craniofacial development in humans and animal models. During zebrafish embryogenesis, twist family members (twist1a, twist1b, twist2, and twist3) function to regulate craniofacial skeletal development. Because of their roles as master regulators of stem cell biology, we hypothesized that twist TFs regulate adult EOM repair and regeneration. In this study, utilizing an adult zebrafish EOM regeneration model, we demonstrate that inhibiting twist3 function using translation-blocking morpholino oligonucleotides (MOs) impairs muscle regeneration by reducing myocyte dedifferentiation and proliferation in the regenerating muscle. This supports our hypothesis that twist TFs are involved in the early steps of dedifferentiation and highlights the importance of twist3 during EOM regeneration.

Paradoxical Changes Underscore Epigenetic Reprogramming During Adult Zebrafish Extraocular Muscle Regeneration.

Purpose: Genomic reprogramming and cellular dedifferentiation are critical to the success of de novo tissue regeneration in lower vertebrates such as zebrafish and axolotl. In tissue regeneration following injury or disease, differentiated cells must retain lineage while assuming a progenitor-like identity in order to repopulate the damaged tissue. Understanding the epigenetic regulation of programmed cellular dedifferentiation provides unique insights into the biology of stem cells and cancer and may lead to novel approaches for treating human degenerative conditions. Methods: Using a zebrafish in vivo model of adult muscle regeneration, we utilized chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) to characterize early changes in epigenetic signals, focusing on three well-studied histone modifications-histone H3 trimethylated at lysine 4 (H3K4me3), and histone H3 trimethylated or acetylated at lysine 27 (H3K27me3 and H3K27Ac, respectively). Results: We discovered that zebrafish myocytes undergo a global, rapid, and transient program to drive genomic remodeling. The timing of these epigenetic changes suggests that genomic reprogramming itself represents a distinct sequence of events, with predetermined checkpoints, to generate cells capable of de novo regeneration. Importantly, we uncovered subsets of genes that maintain epigenetic marks paradoxical to changes in expression, underscoring the complexity of epigenetic reprogramming. Conclusions: Within our model, histone modifications previously associated with gene expression act for the most part as expected, with exceptions suggesting that zebrafish chromatin maintains an easily editable state with a number of genes paradoxically marked for transcriptional activity despite downregulation.