Lupus dermal fibroblasts are proinflammatory and exhibit a profibrotic phenotype in scarring skin disease.

Fibroblasts are stromal cells known to regulate local immune responses important for wound healing and scar formation; however, the cellular mechanisms driving damage and scarring in patients with cutaneous lupus erythematosus (CLE) remain poorly understood. Dermal fibroblasts in patients with systemic lupus erythematosus (SLE) experience increased cytokine signaling in vivo, but the effect of inflammatory mediators on fibroblast responses in nonscarring versus scarring CLE subtypes is unclear. Here, we examined responses to cytokines in dermal fibroblasts from nonlesional skin of 22 patients with SLE and CLE and 34 individuals acting as healthy controls.

Epidermal ZBP1 stabilizes mitochondrial Z-DNA to drive UV-induced IFN signaling in autoimmune photosensitivity.

Photosensitivity is observed in numerous autoimmune diseases and drives poor quality of life and disease flares. Elevated epidermal type I interferon (IFN) production primes for photosensitivity and enhanced inflammation, but the substrates that sustain and amplify this cycle remain undefined. Here, we show that IFN-induced Z-DNA binding protein 1 (ZBP1) stabilizes ultraviolet (UV)B-induced cytosolic Z-DNA derived from oxidized mitochondrial DNA.

Single-cell sequencing reveals Hippo signaling as a driver of fibrosis in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a chronic inflammatory disease characterized by abscesses, nodules, dissecting/draining tunnels, and extensive fibrosis. Here, we integrate single-cell RNA sequencing, spatial transcriptomics, and immunostaining to provide an unprecedented view of the pathogenesis of chronic HS, characterizing the main cellular players and defining their interactions. We found a striking layering of the chronic HS infiltrate and identified the contribution of 2 fibroblast subtypes (SFRP4+ and CXCL13+) in orchestrating this compartmentalized immune response.

Pansclerotic morphea is characterized by IFN-γ responses priming dendritic cell fibroblast crosstalk to promote fibrosis.

Pansclerotic morphea (PSM) is a rare, devastating disease characterized by extensive soft tissue fibrosis, secondary contractions, and significant morbidity. PSM pathogenesis is unknown, and aggressive immunosuppressive treatments rarely slow disease progression. We aimed to characterize molecular mechanisms driving PSM and to identify therapeutically targetable pathways by performing single-cell and spatial RNA-Seq on 7 healthy controls and on lesional and nonlesional skin biopsies of a patient with PSM 12 months apart.

Regulation of Photosensitivity by the Hippo Pathway in Lupus Skin.

Objective: Photosensitivity is one of the most common manifestations of systemic lupus erythematosus (SLE), yet its pathogenesis is not well understood. The normal-appearing epidermis of patients with SLE exhibits increased ultraviolet B (UVB)-driven cell death that persists in cell culture. Here, we investigated the role of epigenetic modification and Hippo signaling in enhanced UVB-induced apoptosis seen in SLE keratinocytes.

Synthesis and biological activity of conformationally restricted indole-based inhibitors of neurotropic alphavirus replication: Generation of a three-dimensional pharmacophore.

We have previously reported the development of indole-based CNS-active antivirals for the treatment of neurotropic alphavirus infection, but further optimization is impeded by a lack of knowledge of the molecular target and binding site. Herein we describe the design, synthesis and evaluation of a series of conformationally restricted analogues with the dual objectives of improving potency/selectivity and identifying the most bioactive conformation. Although this campaign was only modestly successful at improving potency, the sharply defined SAR of the rigid analogs enabled the definition of a three-dimensional pharmacophore, which we believe will be of value in further analog design and virtual screening for alternative antiviral leads.

Flavin-Containing Monooxygenases Are Conserved Regulators of Stress Resistance and Metabolism.

Flavin-Containing Monooxygenases are conserved xenobiotic-detoxifying enzymes. Recent studies have revealed endogenous functions of FMOs in regulating longevity in and in regulating aspects of metabolism in mice. To explore the cellular mechanisms of FMO's endogenous function, here we demonstrate that all five functional mammalian FMOs may play similar endogenous roles to improve resistance to a wide range of toxic stresses in both kidney and liver cells.

Endoplasmic reticulum stress sensor IRE1α propels neutrophil hyperactivity in lupus.

Neutrophils amplify inflammation in lupus through the release of neutrophil extracellular traps (NETs). The endoplasmic reticulum stress sensor inositol-requiring enzyme 1 α (IRE1α) has been implicated as a perpetuator of inflammation in various chronic diseases; however, IRE1α has been little studied in relation to neutrophil function or lupus pathogenesis. Here, we found that neutrophils activated by lupus-derived immune complexes demonstrated markedly increased IRE1α ribonuclease activity.

Staphylococcus aureus Colonization Is Increased on Lupus Skin Lesions and Is Promoted by IFN-Mediated Barrier Disruption.

Cutaneous inflammation is recurrent in systemic lupus erythematosus (SLE), yet mechanisms that drive cutaneous inflammation in SLE are not well defined. Type I IFNs are elevated in nonlesional SLE skin and promote inflammatory responses. Staphylococcus aureus, known to induce IFN production, could play a role in cutaneous inflammation in SLE.

Large-Scale Analysis of Kinase Signaling in Yeast Pseudohyphal Development Identifies Regulation of Ribonucleoprotein Granules.

Yeast pseudohyphal filamentation is a stress-responsive growth transition relevant to processes required for virulence in pathogenic fungi. Pseudohyphal growth is controlled through a regulatory network encompassing conserved MAPK (Ste20p, Ste11p, Ste7p, Kss1p, and Fus3p), protein kinase A (Tpk2p), Elm1p, and Snf1p kinase pathways; however, the scope of these pathways is not fully understood. Here, we implemented quantitative phosphoproteomics to identify each of these signaling networks, generating a kinase-dead mutant in filamentous S.

Discovery of anthranilamides as a novel class of inhibitors of neurotropic alphavirus replication.

Neurotropic alphaviruses are debilitating pathogens that infect the central nervous system (CNS) and are transmitted to humans via mosquitoes. There exist no effective human vaccines against these viruses, underlining the need for effective antivirals, but no antiviral drugs are available for treating infection once the viruses have invaded the CNS. Previously, we reported the development of novel indole-2-carboxamide-based inhibitors of alphavirus replication that demonstrate significant reduction of viral titer and achieve measurable brain permeation in a pharmacokinetic mouse model.

Novel indole-2-carboxamide compounds are potent broad-spectrum antivirals active against western equine encephalitis virus in vivo.

Neurotropic alphaviruses, including western, eastern, and Venezuelan equine encephalitis viruses, cause serious and potentially fatal central nervous system infections in humans for which no currently approved therapies exist. We previously identified a series of thieno[3,2-b]pyrrole derivatives as novel inhibitors of neurotropic alphavirus replication, using a cell-based phenotypic assay (W. Peng et al.

Discovery of potent broad spectrum antivirals derived from marine actinobacteria.

Natural products provide a vast array of chemical structures to explore in the discovery of new medicines. Although secondary metabolites produced by microbes have been developed to treat a variety of diseases, including bacterial and fungal infections, to date there has been limited investigation of natural products with antiviral activity. In this report, we used a phenotypic cell-based replicon assay coupled with an iterative biochemical fractionation process to identify, purify, and characterize antiviral compounds produced by marine microbes.

Optimization of novel indole-2-carboxamide inhibitors of neurotropic alphavirus replication.

Neurotropic alphaviruses, which include western equine encephalitis virus (WEEV) and Fort Morgan virus, are mosquito-borne pathogens that infect the central nervous system causing acute and potentially fatal encephalitis. We previously reported a novel series of indole-2-carboxamides as alphavirus replication inhibitors, one of which conferred protection against neuroadapted Sindbis virus infection in mice. We describe here further development of this series, resulting in 10-fold improvement in potency in a WEEV replicon assay and up to 40-fold increases in half-lives in mouse liver microsomes.

Genetic networks inducing invasive growth in Saccharomyces cerevisiae identified through systematic genome-wide overexpression.

The budding yeast Saccharomyces cerevisiae can respond to nutritional and environmental stress by implementing a morphogenetic program wherein cells elongate and interconnect, forming pseudohyphal filaments. This growth transition has been studied extensively as a model signaling system with similarity to processes of hyphal development that are linked with virulence in related fungal pathogens. Classic studies have identified core pseudohyphal growth signaling modules in yeast; however, the scope of regulatory networks that control yeast filamentation is broad and incompletely defined.

A large-scale complex haploinsufficiency-based genetic interaction screen in Candida albicans: analysis of the RAM network during morphogenesis.

The morphogenetic transition between yeast and filamentous forms of the human fungal pathogen Candida albicans is regulated by a variety of signaling pathways. How these pathways interact to orchestrate morphogenesis, however, has not been as well characterized. To address this question and to identify genes that interact with the Regulation of Ace2 and Morphogenesis (RAM) pathway during filamentation, we report the first large-scale genetic interaction screen in C.

A profile of differentially abundant proteins at the yeast cell periphery during pseudohyphal growth.

Yeast filamentous growth is a stress response to conditions of nitrogen deprivation, wherein yeast colonies form pseudohyphal filaments of elongated and connected cells. As proteins mediating adhesion and transport are required for this growth transition, we expect that the protein complement at the yeast cell periphery plays a critical and tightly regulated role in pseudohyphal filamentation. To identify proteins differentially abundant at the yeast cell periphery during pseudohyphal growth, we generated quantitative proteomic profiles of plasma membrane protein preparations under conditions of vegetative growth and filamentation.

Conditional nuclear import and export of yeast proteins using a chemical inducer of dimerization.

In eukaryotes, reversible shuttling between the nucleus and cytoplasm is an important regulatory mechanism, particularly for many kinases and transcription factors. Inspired by the natural system, we recently developed a technology to control protein position in budding yeast using a chemical inducer of dimerization (CID). In this method, a nuclear export or localization signal is reversibly appended to a protein of interest by the CID, which effectively places its subcellular location under direct control of the chemical stimulus.

A small molecule-directed approach to control protein localization and function.

Protein localization is tightly linked with function, such that the subcellular distribution of a protein serves as an important control point regulating activity. Exploiting this regulatory mechanism, we present here a general approach by which protein location, and hence function, may be controlled on demand in the budding yeast. In this system a small molecule, rapamycin, is used to temporarily recruit a strong cellular address signal to the target protein, placing subcellular localization under control of the selective chemical stimulus.

Localization of autophagy-related proteins in yeast using a versatile plasmid-based resource of fluorescent protein fusions.

Plasmid-based collections of fluorescent protein fusions are valuable and versatile resources, facilitating systematic studies of protein localization in multiple genetic backgrounds. At present, however, few such collections exist for the analysis of protein localization in any organism. To address this deficiency, we present here a plasmid-based set of resources for the analysis of protein localization in the budding yeast.

Analysis of the yeast kinome reveals a network of regulated protein localization during filamentous growth.

The subcellular distribution of kinases and other signaling proteins is regulated in response to cellular cues; however, the extent of this regulation has not been investigated for any gene set in any organism. Here, we present a systematic analysis of protein kinases in the budding yeast, screening for differential localization during filamentous growth. Filamentous growth is an important stress response involving mitogen-activated protein kinase and cAMP-dependent protein kinase signaling modules, wherein yeast cells form interconnected and elongated chains.

Unconventional genomic architecture in the budding yeast saccharomyces cerevisiae masks the nested antisense gene NAG1.

The genomic architecture of the budding yeast Saccharomyces cerevisiae is typical of other eukaryotes in that genes are spatially organized into discrete and nonoverlapping units. Inherent in this organizational model is the assumption that protein-coding sequences do not overlap completely. Here, we present evidence to the contrary, defining a previously overlooked yeast gene, NAG1 (for nested antisense gene) nested entirely within the coding sequence of the YGR031W open reading frame in an antisense orientation on the opposite strand.

Large-scale analysis of yeast filamentous growth by systematic gene disruption and overexpression.

Under certain conditions of nutrient stress, the budding yeast Saccharomyces cerevisiae initiates a striking developmental transition to a filamentous form of growth, resembling developmental transitions required for virulence in closely related pathogenic fungi. In yeast, filamentous growth involves known mitogen-activated protein kinase and protein kinase A signaling modules, but the full scope of this extensive filamentous response has not been delineated. Accordingly, we have undertaken the first systematic gene disruption and overexpression analysis of yeast filamentous growth.

An interrelationship between autophagy and filamentous growth in budding yeast.

Over the last 15 years, yeast pseudohyphal growth (PHG) has been the focus of intense research interest as a model of fungal pathogenicity. Specifically, PHG is a stress response wherein yeast cells deprived of nitrogen form filaments of elongated cells. Nitrogen limitation also induces autophagy, a ubiquitous eukaryotic stress response in which proteins are trafficked to the vacuole/lysosome for degradation and recycling.