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Atopic dermatitis (AD), driven by interleukins (IL‐4/IL‐13), is a chronic inflammatory skin disease characterized by intensive pruritus. However, it is unclear how immune signaling and sensory response pathways cross talk with each other. We differentiated itch sensory neuron‐like cells (ISNLCs) from iPSC lines. These ISNLCs displayed neural markers and action potentials and responded specifically to itch‐specific stimuli. These ISNLCs expressed receptors specific for IL‐4/IL‐13 and were activated directly by the two cytokines. We successfully innervated these ISNLCs into full thickness human skin constructs. These innervated skin grafts can be used in clinical applications such as wound healing. Moreover, the availability of such innervated skin models will be valuable to develop drugs to treat skin diseases such as AD.

Canonical ultraviolet (UV) mutation type and spectra are traditionally defined by direct sequencing-based approaches to map mutations in a limited number of representative DNA elements. To obtain an unbiased view of genome wide UV mutation features, we performed whole exome-sequencing (WES) to profile single nucleotide substitutions in UVB-irradiated primary human keratinocytes. Cross comparison of UV mutation profiles under different UVB radiation conditions revealed that T > C transition was highly prevalent in addition to C > T transition. We also identified 5′-ACG-3′ as a common sequence motif of C > T transition. Furthermore, our analyses uncovered several recurring UV mutations following acute UVB radiation affecting multiple genes including HRNR, TRIOBP, KCNJ12, and KMT2C, which are frequently mutated in skin cancers, indicating their potential role as founding mutations in UV-induced skin tumorigenesis. Pretreatment with trichostatin A, a pan-histone deacetylase inhibitor that renders chromatin decondensation, significantly decreased the number of mutations in UVB-irradiated keratinocytes. Unexpectedly, we found trichostatin A to be a mutagen that caused DNA damage and mutagenesis at least partly through increased reactive oxidation. In summary, our study reveals new UV mutation features following acute UVB radiation and identifies novel UV mutation hotspots that may potentially represent founding driver mutations in skin cancer development.

Solar UV radiation is a major environmental risk factor for skin cancer. Despite decades of robust and meritorious investigation, our understanding of the mechanisms underlying UV-induced skin carcinogenesis remain incomplete. We previously performed comprehensive transcriptomic profiling in human keratinocytes following exposure to different UV radiation conditions to generate UV-specific gene expression signatures. In this study, we utilized Virtual Inference of Protein Activity by Enriched Regulon (VIPER), a robust systems biology tool, on UV-specific skin cell gene signatures to identify master regulators (MRs) of UV-induced transcriptomic changes. We identified multiple prominent candidate UV MRs, including forkhead box M1 (FOXM1), thyroid hormone receptor interactor 13 and DNA isomerase II alpha, which play important roles in cell cycle regulation and genome stability. MR protein activity was either activated or suppressed by UV in normal keratinocytes. Intriguingly, many of the UV-suppressed MRs were activated in human skin squamous cell carcinomas (SCCs), highlighting their importance in skin cancer development. We further demonstrated that selective inhibition of FOXM1, whose activity was elevated in SCC cells, was detrimental to SCC cell survival. Taken together, our study uncovered novel UV MRs that can be explored as new therapeutic targets for future skin cancer treatment.

The lethal toxin (LeTx) of Bacillus anthracis plays a central role in the pathogenesis of anthrax-associated shock. Platelet-activating factor (PAF) is a potent lipid mediator that has been implicated in endotoxin-associated shock. In this study, we examined the contribution of PAF to the manifestations of lethal toxin challenge in WT mice. LeTx challenge resulted in transient increase in serum PAF levels and a concurrent decrease in PAF acetylhydrolase activity. Inhibition of PAF activity using PAF antagonists or toxin challenge of PAF receptor negative mice reversed or ameliorated many of the pathologic features of LeTx-induced damage, including changes in vascular permeability, hepatic necrosis, and cellular apoptosis. In contrast, PAF inhibition had minimal effects on cytokine levels. Findings from these studies support the continued study of PAF antagonists as potential adjunctive agents in the treatment of anthrax-associated shock.

We have reported that TLR11 contributes to the murine response to flagellin and resistance to infection with Salmonella spp., including those responsible for typhoid fever in humans (Mathur et al., 2012). Here, we respond to the Correspondence in this issue of Cell by Song et al. (2016), which claims that mice lacking TLR11 are not more susceptible to infection with Salmonella species and that TLR11 does not recognize Salmonella flagellin.

Toll-like receptors (TLRs) are innate immune receptors that sense a variety of pathogen-associated molecular patterns (PAMPs) by interacting with them and subsequently initiating signal transduction cascades that elicit immune responses. TLR11 has been shown to interact with two known protein PAMPs: Salmonella and E. coli flagellin FliC and Toxoplasma gondii profilin-like protein. Given the highly divergent biology of these pathogens recognized by TLR11, it is unclear whether common mechanisms are used to recognize these distinct protein PAMPs. Here we show that TLR11 interacts with these two PAMPs using different receptor domains. Furthermore, TLR11 binding to flagellin and profilin exhibits differential dependency on pH and receptor ectodomain cleavage.

Naturally occurring mutations in the human RECQ3 gene result in truncated Werner protein (WRN) and manifest as a rare premature aging disorder, Werner syndrome. Cellular and biochemical studies suggest a multifaceted role of WRN in DNA replication, DNA repair, recombination, and telomere maintenance. The RecQ C-terminal (RQC) domain of WRN was determined previously to be the major site of interaction for DNA and proteins. By using site-directed mutagenesis in the WRN RQC domain, we determined which amino acids might be playing a critical role in WRN function. A site-directed mutation at Lys-1016 significantly decreased WRN binding to fork or bubble DNA substrates. Moreover, the Lys-1016 mutation markedly reduced WRN helicase activity on fork, D-loop, and Holliday junction substrates in addition to reducing significantly the ability of WRN to stimulate FEN-1 incision activities. Thus, DNA binding mediated by the RQC domain is crucial for WRN helicase and its coordinated functions. Our nuclear magnetic resonance data on the three-dimensional structure of the wild-type RQC and Lys-1016 mutant proteins display a remarkable similarity in their structures.

Human upstream binding factor (hUBF) is a nucleolar transcription factor involved in transcription by RNA polymerase I. It contains six HMG box domains. The contribution of each HMG box motif to its function is different. hUBF HMG box 1 shows a very strong binding affinity for both the four-way DNA junction and a 15 bp GC-rich rRNA gene core promoter fragment, but hUBF HMG box 5 shows a much weaker binding affinity for the four-way DNA junction and the GC-rich rRNA gene core promoter fragment. To illustrate the molecular basis of their DNA binding difference, the solution structure of box 5 was studied by NMR. The tertiary structure of box 5 shows a common flattened L-shaped fold, similar to box 1 and other HMG boxes with known structures. It is formed by intersection of three helical arms: helix 1 (residues 9-25) and helix 2 (residues 30-42) pack into each other to form the major wing, while helix 3 (residues 48-70) is aligned with the extended N-terminal segment to form the minor wing. A hydrophobic core is formed by three tryptophans (W14, W41, and W52) to maintain the fold. Although there is similarity between the two structures, negative charged electrostatic surface potential in the concave face of the molecule of box 5 exhibits great difference compared to that of box 1 and other HMG boxes with known structures. That surface is involved in DNA binding. Besides, in positions which are involved in intercalating into a DNA base pair, there are hydrophobic residues in box 1 and other HMG boxes but polar residues in box 5. These differences may contribute to the loss of the DNA binding ability of box 5.

The DNA-binding domain of the human transcription factor E2F1 was expressed in Escherichia coli. Through a single purification step using a Ni2+ column, 40-50 mg of the highly purified recombinant protein was obtained from 1 liter of bacterial culture. In addition, it was shown that the recombinant protein had higher stability and solubility under acidic conditions than at a neutral or alkaline pH. The gel shift assay showed that the recombinant E2F1 DNA-binding domain was active in binding a fragment containing E2F sites. Circular dichroism measurements revealed that the recombinant protein approximately contains 33% alpha-helix, 11% beta-sheet, 5% turn, and 51% random coil at pH 7.0, and there was no obvious change for the secondary structure of the recombinant protein between pH 4.0 and pH 9.0. A 3D model was obtained by comparative protein modeling with a homologous protein whose structure was known by program Modeller 4. With the program DSSP, the predicted secondary structure content of the model was consistent with the result of circular dichroism spectrum.

Werner syndrome (WS) is an autosomal recessive disease that results in premature aging. Mutations in the WS gene ( WRN ) result in a loss of expression of the WRN protein and predispose WS patients to accelerated aging. As a helicase and a nuclease, WRN is unique among the five human RecQ helicase family members and is capable of multiple functions involved in DNA replication, repair, recombination, and telomere maintenance. A 144-residue fragment of WRN was previously determined to be a multifunctional DNA- and protein-binding domain (DPBD) that interacts with structure-specific DNA and a variety of DNA-processing proteins. In addition, DPBD functions as a nucleolar targeting sequence of WRN. The solution structure of the DPBD, the first of a WRN fragment, has been solved by NMR. DPBD consists of a winged helix-like motif and an unstructured C-terminal region of ≈20 aa. The putative DNA-binding surface of DPBD has been identified by using known structural and biochemical data. Based on the structural data and on the biochemical data, we suggest a surface on the DPBD for interacting with other proteins. In this structural model, a single winged helix domain binds to both DNA and other proteins. Furthermore, we propose that DPBD functions as a regulatory domain to regulate the enzymatic activity of WRN and to direct cellular localization of WRN through protein–protein interaction.