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Results show that the protein p21 responds to DNA damage and its UV-induced degradation is required for optimal DNA repair. Further, the UV degradation of p21 protein switches to ubiquitin/Skp2-dependent proteasome pathway upon UV irradiation. p21 protein is a cyclin-dependent kinase inhibitor.

Eosinophilic esophagitis (EoE) is an antigen-mediated eosinophilic disease of the esophagus that involves fibroblast activation and progression to fibrostenosis. Cytokines produced by T-helper type 2 cells and transforming growth factor beta 1 (TGFβ1) contribute to the development of EoE, but other cytokines involved in pathogenesis are unknown. We investigate the effects of tumor necrosis factor superfamily member 14 (TNFSF14, also called LIGHT) on fibroblasts in EoE. We analyzed publicly available esophageal CD3+ T-cell single-cell sequencing data for expression of LIGHT. Esophageal tissues were obtained from pediatric patients with EoE or control individuals and analyzed by immunostaining. Human primary esophageal fibroblasts were isolated from esophageal biopsy samples of healthy donors or patients with active EoE. Fibroblasts were cultured; incubated with TGFβ1 and/or LIGHT; and analyzed by RNA sequencing, flow cytometry, immunoblots, immunofluorescence, or reverse transcription polymerase chain reaction. Eosinophils were purified from peripheral blood of healthy donors, incubated with interleukin 5, cocultured with fibroblasts, and analyzed by immunohistochemistry. LIGHT was up-regulated in the esophageal tissues from patients with EoE, compared with control individuals, and expressed by several T-cell populations, including T-helper type 2 cells. TNF receptor superfamily member 14 (TNFRSF14, also called HVEM) and lymphotoxin beta receptor are receptors for LIGHT that were expressed by fibroblasts from healthy donors or patients with active EoE. Stimulation of esophageal fibroblasts with LIGHT induced inflammatory gene transcription, whereas stimulation with TGFβ1 induced transcription of genes associated with a myofibroblast phenotype. Stimulation of fibroblasts with TGFβ1 increased expression of HVEM; subsequent stimulation with LIGHT resulted in their differentiation into cells that express markers of myofibroblasts and inflammatory chemokines and cytokines. Eosinophils tethered to esophageal fibroblasts after LIGHT stimulation via intercellular adhesion molecule-1. T cells in esophageal tissues from patients with EoE express increased levels of LIGHT compared with control individuals, which induces differentiation of fibroblasts into cells with inflammatory characteristics. TGFβ1 increases fibroblast expression of HVEM, a receptor for LIGHT. LIGHT mediates interactions between esophageal fibroblasts and eosinophils via ICAM1. This pathway might be targeted for the treatment of EoE. [ABSTRACT FROM AUTHOR]

Point-of-care (POC) diagnostic biosensors offer a promising solution to improve healthcare, not only in developed parts of the world, but also in resource limited areas that lack adequate medical infrastructure and trained technicians. However, in remote and resource limited settings, cost and storage of traditional POC immunoassays often limit actual deployment. Synthetically engineered biological components (“BioBricks”) provide an avenue to reduce costs and simplify assay procedures. In this article, the design and development of an ultra-low cost, whole-cell “renewable” capture reagent for use in POC diagnostic applications is described. Yeast cells were genetically modified to display both single chain variable fragment (scFv) antibodies and gold-binding peptide (GBP) on their surfaces for simple one step enrichment and surface functionalization. Electrochemical impedance spectroscopy (EIS) and fluorescent imaging were used to verify and characterize the binding of cells to gold electrodes. A complete electrochemical detection assay was then performed on screen-printed electrodes fixed with yeast displaying scFv directed to Salmonella outer membrane protein D (OmpD). Electrochemical assays were optimized and cross-validated with established fluorescence techniques. Nanomolar detection limits were observed for both formats. [ABSTRACT FROM AUTHOR]

Abstract: The mechanism underlying the chemopreventive effects of the non-steroidal anti-inflammatory drug sulindac remains unclear. Its active metabolite, sulindac sulfide, induces cell cycle arrest as well as apoptosis in mammalian cell lines. We now show that in murine thymocytes, sulindac sulfide-induced cell death is p53, bax, Fas, and FasL independent. In contrast, bcl2 transgenic thymocytes are resistant to sulindac sulfide-induced apoptosis. In addition, we demonstrate that sulindac sulfide-induced cell cycle arrest in mouse embryonic fibroblasts (MEFs) is partly mediated by the retinoblastoma tumor suppressor protein (Rb) and the cyclin kinase inhibitor p21waf1/cip1. Furthermore, MEFs deficient in p21 or Rb are more susceptible to sulindac sulfide-induced cell death. These results suggest that sulindac may selectively target premalignant cells with cell cycle checkpoint deficits. [Copyright &y& Elsevier]

The five subunit replication factor C (RF-C) complex plays a critical role in DNA elongation. We find that the large subunit of RF-C (RF-Cp145) is phosphorylated in vivo whereas the smaller RF-C subunits are not phosphorylated. The phosphorylation of endogenous RFCp145 is modulated in a cell cycle-dependent manner. Phosphorylation is maximal in G[sub 2]/M and is inhibited by an inhibitor of cyclin-dependent kinases. Phosphorylation of purified recombinant RF-C complex in vitro reveals that RF-Cp145 is preferentially phosphorylated by cdc2-cyclin B but not by cdk2-cyclin A or cdk2-cyclin E. In vitro phosphorylation of RF-C complex by cdc2-cyclin B kinases leads to dissociation of phosphorylated RF. Cp145 from the RF-C complex. Using different approaches we demonstrate that phosphorylated RFCp145 is indeed dissociated from RF-Cp40 and RF-Cp37 in vivo. These results suggest that destabilization of the RF-C complex by CDKs may inactivate the RF-C complex at the end of S phase. [ABSTRACT FROM AUTHOR]