Chelation of [111In]In3+ with the dual-size-selective macrocycles py-macrodipa and py2-macrodipa.

Indium-111 (111In) is a diagnostic radiometal that is important in nuclear medicine for single-photon emission computed tomography (SPECT). In order to apply this radiometal, it needs to be stably chelated and conjugated to a targeting vector that delivers it to diseased tissue. Identifying effective chelators that are capable of binding and retaining [111In]In3+in vivo is an important research area. In this study, two 18-membered macrocyclic chelators, py-macrodipa and py2-macrodipa, were investigated for their ability to form stable coordination complexes with In3+ and to be effectively radiolabeled with [111In]In3+. The In3+ complexes of these two chelators were characterized by NMR spectroscopy, X-ray crystallography, and density functional theory calculations. These studies show that both py-macrodipa and py2-macrodipa form 8-coordinate In3+ complexes and attain an asymmetric conformation, consistent with prior studies on this ligand class with small rare earth metal ions. Spectrophotometric titrations were carried out to determine the thermodynamic stability constants (log KML) of [In(py-macrodipa)]+ and [In(py2-macrodipa)]+, which were found to be 18.96(6) and 19.53(5), respectively, where the values in parentheses are the errors of the last significant figures obtained from the standard deviation from three independent replicates. Radiolabeling studies showed that py-macrodipa and py2-macrodipa can quantitatively be radiolabeled with [111In]In3+ at 25 °C within 5 min, even at ligand concentrations as low as 1 µM. The in vitro stability of the radiolabeled complexes was investigated in human serum at 37 °C, revealing that ∼90% of [111In][In(py-macrodipa)]+ and [111In][In(py2-macrodipa)]+ remained intact after 7 days. The biodistribution of these radiolabeled complexes in mice was investigated, showing lower uptake in the kidneys, liver, and blood at the 24 h mark compared to [111In]InCl3. These results demonstrate the potential of py-macrodipa and py2-macrodipa as chelators for [111In]In3+, suggesting their value for SPECT radiopharmaceuticals.

Integrated plasma metabolomic and cytokine analysis reveals a distinct immunometabolic signature in atopic dermatitis.

Importance: Disease models for atopic dermatitis (AD) have primarily focused on understanding underlying environmental, immunologic, and genetic etiologies. However, the role of metabolic mechanisms in AD remains understudied. Objective: To investigate the circulating blood metabolomic and cytokine profile of AD as compared to healthy control patients. Design: This study collected plasma from 20 atopic dermatitis with moderate-to-severe itch (score of ≥5 on the itch Numeric Rating Scale and IGA score ≥3) and 24 healthy control patients. Mass-spectrometry based metabolite data were compared between AD and healthy controls. Unsupervised and supervised machine learning algorithms and univariate analysis analyzed metabolic concentrations. Metabolite enrichment and pathway analyses were performed on metabolites with significant fold change between AD and healthy control patients. To investigate the correlation between metabolites levels and cytokines, Spearman's rank correlation coefficients were calculated between metabolites and cytokines. Setting: Patients were recruited from the Johns Hopkins Itch Center and dermatology outpatient clinics in the Johns Hopkins Outpatient Center. Participants: The study included 20 atopic dermatitis patients and 24 healthy control patients. Main outcomes and measures: Fold changes of metabolites in AD vs healthy control plasma. Results: In patients with AD, amino acids isoleucine, tyrosine, threonine, tryptophan, valine, methionine, and phenylalanine, the amino acid derivatives creatinine, indole-3-acrylic acid, acetyl-L-carnitine, L-carnitine, 2-hydroxycinnamic acid, N-acetylaspartic acid, and the fatty amide oleamide had greater than 2-fold decrease (all P-values<0.0001) compared to healthy controls. Enriched metabolites were involved in branched-chain amino acid (valine, leucine, and isoleucine) degradation, catecholamine biosynthesis, thyroid hormone synthesis, threonine metabolism, and branched and long-chain fatty acid metabolism. Dysregulated metabolites in AD were positively correlated cytokines TARC and MCP-4 and negatively correlated with IL-1a and CCL20. Conclusions and relevance: Our study characterized novel dysregulated circulating plasma metabolites and metabolic pathways that may be involved in the pathogenesis of AD. These metabolic pathways serve as potential future biomarkers and therapeutic targets in the treatment of AD.

Dysregulation of the Skin-Liver Axis in Prurigo Nodularis: An Integrated Genomic, Transcriptomic, and Population-Based Analysis.

Pruritus has long been linked to hepatic dysfunction; however, there are limited data characterizing the association between liver disease and prurigo nodularis (PN), a chronic inflammatory skin disease featuring severe pruritis. We thus conducted a cross-sectional analysis of hepatic comorbidities in PN patients using TriNetX, a large global health research network. This analysis revealed that PN patients had a higher risk (p < 0.001) of developing liver cirrhosis, acute and subacute hepatic failure, inflammatory liver disease, chronic hepatitis, nonalcoholic steatohepatitis, portal hypertension, fatty liver, chronic passive congestion of the liver, and hepatocellular carcinoma compared with healthy controls. The cumulative incidence of liver disease was about three times higher in PN patients compared with healthy controls. These findings provided the basis for translational studies to investigate a genetic mechanism for this association. Cutaneous transcriptomic analysis performed on PN patients revealed the dysregulation of genes related to hepatic failure in lesional PN compared with both nonlesional PN and control skin. Similarly, gene set variation analysis (GSVA) revealed a significantly increased (p < 0.05) activation of liver metabolism, chronic hepatic failure, acute hepatic failure, cholestatic liver disease, polycystic liver disease, and hepatocellular carcinoma pathways in lesional PN compared with control skin. A subsequent genome-wide association study (GWAS) identified shared single-nucleotide polymorphisms (SNPs) in the genes AR, EDIL3, MACROD2, PCSK5, RUNX1T1, TENM4, and ZEB2 between PN and liver disease from the FinnGen cohort. Significant dysregulation of the skin-liver axis in PN patients may explain the increased incidence and severity of hepatic comorbidities and help identify future therapeutic targets for PN.

Spatial Mass Cytometry-Based Single-Cell Imaging Reveals a Disrupted Epithelial-Immune Axis in Prurigo Nodularis.

Prurigo nodularis (PN) is a chronic, inflammatory skin condition that disproportionately affects African Americans and features intensely pruritic, hyperkeratotic nodules on the extremities and trunk. PN is understudied compared with other inflammatory skin diseases, with the spatial organization of the cutaneous infiltrate in PN yet to be characterized. In this work, we employ spatial imaging mass cytometry to visualize PN lesional skin inflammation and architecture with single-cell resolution through an unbiased machine learning approach. PN lesional skin has increased expression of caspase 3, NF-kB, and phosphorylated signal transducer and activator of transcription 3 compared with healthy skin. Keratinocytes in lesional skin are subdivided into CD14+CD33+, CD11c+, CD63+, and caspase 3-positive innate subpopulations. CD14+ macrophage populations expressing phosphorylated extracellular signal-regulated kinase 1/2 correlate positively with patient-reported itch (P = .006). Hierarchical clustering reveals a cluster of patients with PN with greater atopy, increased NF-kB+ signal transducer and activator of transcription 3-positive phosphorylated extracellular signal-regulated kinase 1/2-positive monocyte-derived myeloid dendritic cells, and increased vimentin expression (P < .05). Neighborhood analysis finds interactions between CD14+ macrophages, CD3+ T cells, monocyte-derived myeloid dendritic cells, and keratinocytes expressing innate immune markers. These findings highlight phosphorylated extracellular signal-regulated kinase-positive CD14+ macrophages as contributors to itch and suggest an epithelial-immune axis in PN pathogenesis.

Plasma metabolomic profiling reveals a novel circulating biomarker signature in chronic pruritus of unknown origin.

Chronic pruritus of unknown origin (CPUO) is characterized by chronic itch for 6 weeks or greater without an identifiable primary cause. Studies are needed to investigate circulating blood biomarkers to elucidate disease pathogenesis. The objective of this study was to investigate changes in circulating blood metabolites in CPUO patients and to identify potential therapeutic targets. Our cross-sectional study collected plasma from 11 CPUO patients and 11 matched control patients for mass-spectrometry based metabolite data analysis. 15 metabolites differed significantly in the blood of CPUO patients compared to controls, including nine amino acids (isoleucine, L-tyrosine, threonine, DL-tryptophan, L-valine, methionine, glycine, lysine, and L-phenylalanine), four amino acid derivatives (creatinine, DL-carnitine, acetyl-L-carnitine, and indole-3-acrylic acid), and two aromatic and fatty acid derivatives (2-hydroxycinnamic acid and oleamide). These metabolites were also correlated with itch severity. Metabolic set enrichment analysis (MSEA) identified downregulation of several pathways in CPUO: phenylalanine, tyrosine, tryptophan biosynthesis; catecholamine biosynthesis; and glycine, serine, and threonine metabolism. Our study identified decreases in several circulating plasma metabolites in CPUO patients and downregulation of pathways related to catecholamine biosynthesis and tryptophan biosynthesis, providing insight into the pathogenesis of CPUO.

Efficacy and Safety of Abrocitinib in Prurigo Nodularis and Chronic Pruritus of Unknown Origin: A Nonrandomized Controlled Trial.

Importance: Prurigo nodularis (PN) and chronic pruritus of unknown origin (CPUO) are chronic pruritic diseases that dramatically impair quality of life, but therapeutic options are limited. Abrocitinib, a Janus kinase 1 inhibitor, represents a promising therapy for both conditions. Objective: To assess the efficacy and safety of 200-mg oral abrocitinib administered once daily in adults with moderate to severe PN or CPUO. Design, Setting, and Participants: This phase 2, open-label, nonrandomized controlled trial conducted between September 2021 and July 2022 took place at a single center in the US. A total of 25 adult patients with moderate to severe PN or CPUO were screened. Ten patients with PN and 10 patients with CPUO were enrolled. All 20 patients completed the 12-week treatment period, 18 of whom completed the 4-week follow-up period. Intervention: Abrocitinib, 200 mg, by mouth once daily for 12 weeks. Main Outcomes and Measures: The primary efficacy end point was the percent change in weekly Peak Pruritus Numerical Rating Scale (PP-NRS) scores from baseline to week 12. Key secondary end points included the percentage of patients achieving at least a 4-point reduction in weekly PP-NRS score from baseline to week 12 and the percent change in Dermatology Life Quality Index (DLQI) scores. Results: A total of 10 patients with PN (mean [SD] age, 58.6 [13.1] years; all were female) and 10 patients with CPUO (mean [SD] age, 70.7 [5.6] years; 2 were female) enrolled in the study. The mean (SD) baseline PP-NRS score was 9.2 (1.0) for PN and 8.2 (1.2) for CPUO. PP-NRS scores decreased by 78.3% in PN (95% CI, -118.5 to -38.1; P < .001) and 53.7% in CPUO (95% CI, -98.8 to -8.6; P = .01) by week 12. From baseline to week 12, 8 of 10 patients with PN and 6 of 10 patients with CPUO achieved at least a 4-point improvement on the PP-NRS. Both groups experienced significant improvement in quality of life as demonstrated by percent change in DLQI scores (PN: -53.2% [95% CI, -75.3% to -31.1%]; P = .002; CPUO: -49.0% [95% CI, -89.6% to -8.0%]; P = .02). The most common adverse event among patients was acneiform eruption in 2 of 20 patients (10%). No serious adverse events occurred. Conclusions and Relevance: The results of this nonrandomized controlled trial suggest that abrocitinib monotherapy may be effective and tolerated well in adults with PN or CPUO. Randomized, double-blind, placebo-controlled trials are warranted to validate these findings. Trial Registration: ClinicalTrials.gov Identifier: NCT05038982.

Immune stimulus exposure as a trigger for the development of chronic pruritus and circulating blood type 2 inflammation.

Background: Chronic pruritus (CP) is a poorly characterized condition associated with intense pruritus without a primary skin eruption. This condition tends to emerge more commonly in older adults, and there is limited research on triggering factors. Objective: To explore the clinical characteristics and pathophysiology of CP following exposure to an immune stimulus. Methods: Clinical characteristics and plasma samples were collected from 15 patients who developed CP following an immune stimulus such as checkpoint inhibitors or vaccination. A multiplex panel was used to analyze plasma cytokine concentrations within these patients. Results: Most immunotherapy-treated patients experienced CP during treatment or after 21 to 60 days of receiving treatment, while vaccine-stimulated patients developed pruritus within a week of vaccination. Plasma cytokine analysis revealed elevated levels of 12 cytokines in patients with immune-stimulated CP compared to healthy controls. Notably, T helper 2 (Th2) related cytokines interleukin (IL)-5 (fold change 2.65; q < 0.25) and thymic stromal lymphopoietin (fold change 1.61 q < 0.25) were upregulated. Limitations: Limitations of this study include limited sample size, particularly in the plasma cytokine assay. Conclusions and Relevance: This study reveals triggers of CP development and describes alterations in blood Th2 markers in patients with CP, including IgE, increased blood eosinophils, and cytokines IL-5 and thymic stromal lymphopoietin.

Comprehensive plasma cytokine and chemokine profiling in prurigo nodularis reveals endotypes in Type 2 inflammation.

Prurigo nodularis (PN) is a chronic inflammatory skin disease that is associated with variability in peripheral blood eosinophil levels and response to T-helper 2 targeted therapies (Th2). Our objective was to determine whether circulating immune profiles with respect to type 2 inflammation differ by race and peripheral blood eosinophil count. Plasma from 56 PN patients and 13 matched healthy controls was assayed for 54 inflammatory biomarkers. We compared biomarker levels between PN and HCs, among PN patients based on absolute eosinophil count, and across racial groups in PN. Eleven biomarkers were elevated in PN versus HCs including interleukin (IL)-12/IL-23p40, tumor necrosis factor-alpha (TNF-α), Thymic stromal lymphopoietin (TSLP), and macrophage-derived chemokine (MDC/CCL22). Additionally, PN patients with AEC > 0.3 K cells/µL had higher Th2 markers (eotaxin, eotaxin-3, TSLP, MCP-4/CCL13), and African American PN patients had lower eosinophils, eotaxin, and eotaxin-3 versus Caucasian and Asian PN patients (p < 0.05 for all). Dupilumab responders had higher AEC (p < 0.01), were more likely to be Caucasian (p = 0.02) or Asian (p = 0.05) compared to African Americans, and more often had a history of atopy (p = 0.08). This study suggests that blood AEC > 0.3 K and Asian and Caucasian races are associated with Th2 skewed circulating immune profiles and response to Th2 targeted therapies.

Single-Cell RNA Sequencing Reveals Dysregulated POSTN+WNT5A+ Fibroblast Subclusters in Prurigo Nodularis.

Prurigo nodularis (PN) is an intensely pruritic, inflammatory skin disease with a poorly understood pathogenesis. We performed single-cell transcriptomic profiling of 28,695 lesional and nonlesional PN cells. Lesional PN has increased dysregulated fibroblasts (FBs) and myofibroblasts. FBs in lesional PN were shifted toward a cancer-associated FB-like phenotype, with POSTN+WNT5A+ cancer-associated FBs increased in PN and similarly so in squamous cell carcinoma. A multicenter cohort study revealed an increased risk of squamous cell carcinoma and cancer-associated FB-associated malignancies (breast and colorectal) in patients with PN. Systemic fibroproliferative diseases (renal sclerosis and idiopathic pulmonary fibrosis) were upregulated in patients with PN. Ligand-receptor analyses demonstrated an FB neuronal axis with FB-derived WNT5A and periostin interactions with neuronal receptors melanoma cell adhesion molecule and ITGAV. These findings identify a pathogenic and targetable POSTN+WNT5A+ FB subpopulation that may predispose cancer-associated FB-associated malignancies in patients with PN.

Sequential onset of bilateral trigeminal neuralgia: clinical presentation and outcomes.

PURPOSE: Sequential onset of bilateral trigeminal neuralgia (TN) is rare and not well-described in the literature. The objective of this study was to characterize demographic, clinical, and procedural characteristics of patients with sequential onset bilateral TN. METHODS: We retrospectively reviewed patients presenting with sequential onset bilateral TN at our institution from 2007 to 2020. Patient demographics, clinical diagnoses, pain outcomes, and procedural characteristics were recorded and compared. Factors associated with pain recurrence were assessed using survival analyses and multivariate regressions. RESULTS: We identified 34 patients who presented with sequential onset bilateral TN. The average age of onset for the index case was 49.9 ± 15.5 years, and 58.0 ± 16.8 years for the contralateral case. In total for our cohort, 91 surgical procedures were performed for the index case, and 70 for the contralateral case. With each additional surgical intervention, pain-free survival was more likely to decrease, p = 0.05. When controlled for order of intervention, glycerin rhizotomy (p = 0.01) and glycerin-radiofrequency rhizotomy (p = 0.05) were more likely associated with pain recurrence compared to microvascular decompression. While pain outcomes were significantly decreased in our cohort at final follow-up, 82.4% of patients were still dependent on medication for pain management after an average of 5.03 ± 7.74 years. CONCLUSION: Our results represent one of the largest series of sequential onset bilateral TN in North America. Our study demonstrates the high treatment burden and chronicity of pain encountered in this population.

Single-cell RNA sequencing reveals dysregulated fibroblast subclusters in prurigo nodularis.

Prurigo nodularis (PN) is an intensely pruritic, chronic inflammatory skin disease that disproportionately affects black patients. However, the pathogenesis of PN is poorly understood. We performed single-cell transcriptomic profiling, ligand receptor analysis and cell trajectory analysis of 28,695 lesional and non-lesional PN skin cells to uncover disease-identifying cell compositions and genetic characteristics. We uncovered a dysregulated role for fibroblasts (FBs) and myofibroblasts as a key pathogenic element in PN, which were significantly increased in PN lesional skin. We defined seven unique subclusters of FBs in PN skin and observed a shift of PN lesional FBs towards a cancer-associated fibroblast (CAF)-like phenotype, with WNT5A+ CAFs increased in the skin of PN patients and similarly so in squamous cell carcinoma (SCC). A multicenter PN cohort study subsequently revealed an increased risk of SCC as well as additional CAF-associated malignancies in PN patients, including breast and colorectal cancers. Systemic fibroproliferative diseases were also upregulated in PN patients, including renal sclerosis and idiopathic pulmonary fibrosis. Ligand receptor analyses demonstrated increased FB1-derived WNT5A and periostin interactions with neuronal receptors MCAM and ITGAV, suggesting a fibroblast-neuronal axis in PN. Type I IFN responses in immune cells and increased angiogenesis/permeability in endothelial cells were also observed. As compared to atopic dermatitis (AD) and psoriasis (PSO) patients, increased mesenchymal dysregulation is unique to PN with an intermediate Th2/Th17 phenotype between atopic dermatitis and psoriasis. These findings identify a pathogenic role for CAFs in PN, including a novel targetable WNT5A+ fibroblast subpopulation and CAF-associated malignancies in PN patients.

A Polygenic Risk Score for Predicting Racial and Genetic Susceptibility to Prurigo Nodularis.

Prurigo nodularis (PN) is an understudied inflammatory skin disease characterized by pruritic, hyperkeratotic nodules. Identifying the genetic factors underlying PN could help to better understand its etiology and guide the development of therapies. In this study, we developed a polygenic risk score that predicts a diagnosis of PN (OR = 1.41, P = 1.6 × 10-5) in two independent and continentally distinct populations. We also performed GWASs, which uncovered genetic variants associated with PN, including one near PLCB4 (rs6039266: OR = 3.15, P = 4.8 × 10-8) and others near TXNRD1 (rs34217906: OR = 1.71, P = 6.4 × 10-7; rs7134193: OR = 1.57, P = 1.1 × 10-6). Finally, we discovered that Black patients have over a two-times greater genetic risk of developing PN (OR = 2.63, P = 7.8 × 10-4). Combining the polygenic risk score and self-reported race together was significantly predictive of PN (OR = 1.32, P = 4.7 × 10-3). Strikingly, this association was more significant with race than after adjusting for genetic ancestry. Because race is a sociocultural construct and not a genetically bound category, our findings suggest that genetics, environmental influence, and social determinants of health likely affect the development of PN and may contribute to clinically observed racial disparities.

Risk of Hematologic Cancer in Patients With Undifferentiated Pruritus.

Importance: Although pruritus is common in patients with hematologic cancers, it is unknown whether patients with undifferentiated pruritus have higher risk of developing hematologic cancer. Furthermore, it is unclear whether serum lactate dehydrogenase (LDH) level, commonly ordered for cancer workup, has diagnostic utility in patients with pruritus. Objective: To assess the risk of hematologic cancer and the diagnostic utility of LDH level in patients with undifferentiated pruritus. Design, Setting, and Participants: This retrospective population-level cohort analysis was conducted using the TriNetX Research Network, a global health records database encompassing more than 69 million patients, from 2002 to 2020. The study included 327 502 eligible patients diagnosed with unspecified pruritus, excluding those with existing chronic pruritic dermatoses or systemic diseases known to cause pruritus, along with 327 502 matched controls. Exposures: Development of hematologic cancer within 1 year, 5 years, and 10 years following unspecified pruritus diagnosis. Main Outcomes and Measures: Primary study outcomes were 1-year, 5-year, and 10-year relative risks (RRs) for development of 9 hematologic cancers in patients with pruritus compared with control patients. Secondary outcomes were 1-year, 5-year, and 10-year RRs for any hematologic cancer at different LDH cutoffs (250 U/L and 500 U/L). Results: After matching, the pruritus and control cohorts each had 327 502 patients (68.1% female patients; 0.4% American Indian or Alaska Native patients; 3.5% Asian patients; 22.2% Black patients; 0.1% Native Hawaiian or Pacific Islander patients; 59.3% White patients; mean [SD] age, 42.2 [22] years). Patients with pruritus had increased 1-year risk of Hodgkin lymphoma (RR, 4.42; 95% CI, 2.83-6.88), myeloid leukemia (RR, 2.56; 95% CI, 1.79-3.67), multiple myeloma (RR, 2.38; 95% CI, 1.66-3.41), non-Hodgkin lymphoma (RR, 2.35; 95% CI, 1.96-2.82), monoclonal gammopathy (RR, 1.90; 95% CI, 1.55-2.32), myelodysplastic syndrome (RR, 1.74; 95% CI, 1.14-2.64), and lymphocytic leukemia (RR, 1.47; 95% CI, 1.07-2.02). After 12 months, the cancer risk was comparable with that of controls. Patients with pruritus had increased LDH levels, which were not associated with increased hematologic cancer risk. Conclusions and Relevance: In this cohort study, the RR of hematologic cancer in patients with undifferentiated pruritus was highest in the first 12 months, and LDH level had limited diagnostic utility in these patients. Clinicians should consider a thorough review of symptoms and assessment of cancer risk factors when deciding on workup for patients presenting with undifferentiated pruritus.

Racial differences in dysregulation of the renin-angiotensin-aldosterone system in patients with prurigo nodularis.

BACKGROUND: There are limited data characterizing the association between renal disease and prurigo nodularis (PN). OBJECTIVE: To characterize the association and describe mechanistic disease linkages between PN and renal comorbidities. METHODS: We conducted a cross-sectional analysis of renal comorbidities in PN using TriNetX, a global health research network providing access to medical records. Epidemiological findings provided the basis for translational studies on plasma and skin biopsy samples from PN patients stratified by race. RESULTS: PN was associated with stages 1-5 of chronic kidney disease (CKD), end-stage renal disease, nephritic syndrome, nephrotic syndrome, glomerular disease, and tubulointerstitial disease, but the associations were significantly stronger in black patients. Compared to controls, CKD progression was faster (HR 2.88, 95% CI: 1.01-8.26) only in black PN (10-year survival: 63.5% black vs. 85.5% white). Circulating plasma angiotensinogen levels were dysregulated (P < 0.001) only in black PN patients. Cutaneous transcriptomic analysis of genes related to the renin-angiotensin-aldosterone system (RAAS) revealed considerable dysregulation in PN lesions with greater dysregulation in black patients. CONCLUSIONS: Significant dysregulation of the cutaneous and systemic RAAS in black PN patients may explain the increased incidence and severity of renal disease.

Mouse Models of Syndromic Craniosynostosis.

Craniosynostosis is a common craniofacial birth defect. This review focusses on the advances that have been achieved through studying the pathogenesis of craniosynostosis using mouse models. Classic methods of gene targeting which generate individual gene knockout models have successfully identified numerous genes required for normal development of the skull bones and sutures. However, the study of syndromic craniosynostosis has largely benefited from the production of knockin models that precisely mimic human mutations. These have allowed the detailed investigation of downstream events at the cellular and molecular level following otherwise unpredictable gain-of-function effects. This has greatly enhanced our understanding of the pathogenesis of this disease and has the potential to translate into improvement of the clinical management of this condition in the future.

Malignant mesothelioma cells are rapidly sensitized to TRAIL-induced apoptosis by low-dose anisomycin via Bim.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) holds promise for the treatment of tumors; however, many tumors are resistant to TRAIL alone. We previously showed that resistant malignant mesothelioma cells are sensitized to TRAIL-induced apoptosis by diverse toxic insults including chemotherapy, irradiation, or protein translation inhibitors such as cycloheximide. In seeking nontoxic sensitizers for TRAIL, we tested the protein translation inhibitor anisomycin at subtoxic concentrations 10- to 100-fold below those reported to inhibit protein translation. At these low concentrations (25 ng/mL), anisomycin potently and rapidly sensitized mesothelioma cells to TRAIL-induced apoptosis. Moreover, such sensitization occurred in malignant but not in nonmalignant mesothelial cells. Sensitization by anisomycin was dependent on Bid, indicating a role for mitochondrial amplification in the apoptotic synergy with TRAIL signaling. Consistent with this, we found that anisomycin induces rapid accumulation of the BH3-only protein Bim; moreover, small interfering RNA knockdown of Bim inhibits anisomycin-induced sensitization. Bim accumulation seems not to be transcriptional; instead, it is associated with Bim phosphorylation and increased stability, both consistent with the activation of c-jun NH2-terminal kinase signals by anisomycin. Overall, our data indicate that the rapid and selective sensitization by anisomycin in mesothelioma cells is mediated by posttranslational potentiation of Bim, which primes the cells for apoptosis via the death receptor pathway. Such subtoxic approaches to sensitization may enhance the value of TRAIL in cancer therapy.

The RNA-binding protein YBX1 regulates epidermal progenitors at a posttranscriptional level.

The integrity of stratified epithelia depends on the ability of progenitor cells to maintain a balance between proliferation and differentiation. While much is known about the transcriptional pathways underlying progenitor cells' behavior in the epidermis, the role of posttranscriptional regulation by mRNA binding proteins-a rate-limiting step in sculpting the proteome-remains poorly understood. Here we report that the RNA binding protein YBX1 (Y-box binding protein-1) is a critical effector of progenitors' function in the epidermis. YBX1 expression is restricted to the cycling keratinocyte progenitors in vivo and its genetic ablation leads to defects in the architecture of the skin. We further demonstrate that YBX1 negatively controls epidermal progenitor senescence by regulating the translation of a senescence-associated subset of cytokine mRNAs via their 3' untranslated regions. Our study establishes YBX1 as a posttranscriptional effector required for maintenance of epidermal homeostasis.

Overexpression of Fgfr2c causes craniofacial bone hypoplasia and ameliorates craniosynostosis in the Crouzon mouse.

FGFR2c regulates many aspects of craniofacial and skeletal development. Mutations in the FGFR2 gene are causative of multiple forms of syndromic craniosynostosis, including Crouzon syndrome. Paradoxically, mouse studies have shown that the activation (Fgfr2cC342Y; a mouse model for human Crouzon syndrome), as well as the removal (Fgfr2cnull), of the FGFR2c isoform can drive suture abolishment. This study aims to address the downstream effects of pathogenic FGFR2c signalling by studying the effects of Fgfr2c overexpression. Conditional overexpression of Fgfr2c (R26RFgfr2c;ßact) results in craniofacial hypoplasia as well as microtia and cleft palate. Contrary to Fgfr2cnull and Fgfr2cC342Y, Fgfr2c overexpression is insufficient to drive onset of craniosynostosis. Examination of the MAPK/ERK pathway in the embryonic sutures of Fgfr2cC342Y and R26RFgfr2c;ßact mice reveals that both mutants have increased pERK expression. The contrasting phenotypes between Fgfr2cC342Y and R26RFgfr2c;ßact mice prompted us to assess the impact of the Fgfr2c overexpression allele on the Crouzon mouse (Fgfr2cC342Y), in particular its effects on the coronal suture. Our results demonstrate that Fgfr2c overexpression is sufficient to partially rescue craniosynostosis through increased proliferation and reduced osteogenic activity in E18.5 Fgfr2cC342Y embryos. This study demonstrates the intricate balance of FGF signalling required for correct calvarial bone and suture morphogenesis, and that increasing the expression of the wild-type FGFR2c isoform could be a way to prevent or delay craniosynostosis progression.