Search

Abstract Background Domains are the building blocks of proteins. During evolution, they have been duplicated, fused and recombined, to produce proteins with novel structures and functions. Structural and genome-scale studies have shown that pairs or groups of domains observed together in a protein are almost always found in only one N to C terminal order and are the result of a single recombination event that has been propagated by duplication of the multi-domain unit. Previous studies of domain organisation have used graph theory to represent the co-occurrence of domains within proteins. We build on this approach by adding directionality to the graphs and connecting nodes based on their relative order in the protein. Most of the time, the linear order of domains is conserved. However, using the directed graph representation we have identified non-linear features of domain organization that are over-represented in genomes. Recognising these patterns and unravelling how they have arisen may allow us to understand the functional relationships between domains and understand how the protein repertoire has evolved. Results We identify groups of domains that are not linearly conserved, but instead have been shuffled during evolution so that they occur in multiple different orders. We consider 192 genomes across all three kingdoms of life and use domain and protein annotation to understand their functional significance. To identify these features and assess their statistical significance, we represent the linear order of domains in proteins as a directed graph and apply graph theoretical methods. We describe two higher-order patterns of domain organisation: clusters and bi-directionally associated domain pairs and explore their functional importance and phylogenetic conservation. Conclusion Taking into account the order of domains, we have derived a novel picture of global protein organization. We found that all genomes have a higher than expected degree of clustering and more domain pairs in forward and reverse orientation in different proteins relative to random graphs with identical degree distributions. While these features were statistically over-represented, they are still fairly rare. Looking in detail at the proteins involved, we found strong functional relationships within each cluster. In addition, the domains tended to be involved in protein-protein interaction and are able to function as independent structural units. A particularly striking example was the human Jak-STAT signalling pathway which makes use of a set of domains in a range of orders and orientations to provide nuanced signaling functionality. This illustrated the importance of functional and structural constraints (or lack thereof) on domain organisation.

DNA i-motif structures are formed in the nuclei of human cells and are believed to provide critical genomic regulation. While the existence, abundance, and distribution of i-motif structures in human cells has been demonstrated and studied by immunofluorescent staining, and more recently NMR and CUT&Tag, the abundance and distribution of such structures in human genomic DNA have remained unclear. Here we utilise high-affinity i-motif immunoprecipitation followed by sequencing to map i-motifs in the purified genomic DNA of human MCF7, U2OS and HEK293T cells. Validated by biolayer interferometry and circular dichroism spectroscopy, our approach aimed to identify DNA sequences capable of i-motif formation on a genome-wide scale, revealing that such sequences are widely distributed throughout the human genome and are common in genes upregulated in G0/G1 cell cycle phases. Our findings provide experimental evidence for the widespread formation of i-motif structures in human genomic DNA and a foundational resource for future studies of their genomic, structural, and molecular roles., (© 2024. The Author(s).)

Purpose: We investigated the contribution of genomic data reanalysis to the diagnostic yield of dystonia patients who remained undiagnosed after prior genome sequencing., Methods: Probands with heterogeneous dystonia phenotypes who underwent initial genome sequencing (GS) analysis in 2019 were included in the reanalysis, which was performed through gene-specific discovery collaborations and systematic genomic data reanalysis., Results: Initial GS analysis in 2019 (n = 111) identified a molecular diagnosis in 11.7 % (13/111) of cases. Reanalysis between 2020 and 2023 increased the diagnostic yield by 7.2 % (8/111); 3.6 % (4/111) through focused gene-specific clinical correlation collaborative efforts [VPS16 (two probands), AOPEP and POLG], and 3.6 % (4/111) by systematic reanalysis completed in 2023 [NUS1 (two probands) and DDX3X variants, and a microdeletion encompassing VPS16]. Seven of these patients had a high phenotype-based dystonia score ≥3. Notable unverified findings in four additional cases included suspicious variants of uncertain significance in FBXL4 and EIF2AK2, and potential phenotypic expansion associated with SLC2A1 and TREX1 variants., Conclusion: GS data reanalysis increased the diagnostic yield from 11.7 % to 18.9 %, with potential extension up to 22.5 %. While optimal timing for diagnostic reanalysis remains to be determined, this study demonstrates that periodic re-interrogation of dystonia GS datasets can provide additional genetic diagnoses, which may have significant implications for patients and their families., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kishore Raj Kumar reports financial support was provided by the Ainsworth 4 Foundation. Stephen Tisch reports a relationship with Medtronic that includes: consulting or advisory and speaking and lecture fees. Stephen Tisch reports a relationship with Boston Scientific that includes: consulting or advisory and speaking and lecture fees. Stephen Tisch reports a relationship with Seqirus Pharmaceuticals that includes: consulting or advisory and speaking and lecture fees. Carolyn M. Sue reports a relationship with Living Cell Technologies Ltd. that includes: consulting or advisory. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The expression of genes encompasses their transcription into mRNA followed by translation into protein. In recent years, next-generation sequencing and mass spectrometry methods have profiled DNA, RNA and protein abundance in cells. However, there are currently no reference standards that are compatible across these genomic, transcriptomic and proteomic methods, and provide an integrated measure of gene expression. Here, we use synthetic biology principles to engineer a multi-omics control, termed pREF, that can act as a universal molecular standard for next-generation sequencing and mass spectrometry methods. The pREF sequence encodes 21 synthetic genes that can be in vitro transcribed into spike-in mRNA controls, and in vitro translated to generate matched protein controls. The synthetic genes provide qualitative controls that can measure sensitivity and quantitative accuracy of DNA, RNA and peptide detection. We demonstrate the use of pREF in metagenome DNA sequencing and RNA sequencing experiments and evaluate the quantification of proteins using mass spectrometry. Unlike previous spike-in controls, pREF can be independently propagated and the synthetic mRNA and protein controls can be sustainably prepared by recipient laboratories using common molecular biology techniques. Together, this provides a universal synthetic standard able to integrate genomic, transcriptomic and proteomic methods., (© 2024. The Author(s).)

Hepatoblastoma is characterized by driver mutations in CTNNB1 , making it an attractive biomarker for a liquid biopsy approach utilizing circulating tumor DNA (ctDNA). This prospective observational study sought to ascertain the feasibility of ctDNA detection in patients with hepatoblastoma and explore its associations with established clinical indicators and biomarkers, including serum Alpha-fetoprotein (AFP). We obtained 38 plasma samples and 17 tumor samples from 20 patients with hepatoblastoma. These samples were collected at various stages: 10 at initial diagnosis, 17 during neoadjuvant chemotherapy, 6 post-operatively, and 5 at disease recurrence. Utilizing a bespoke sequencing assay we developed called QUENCH, we identified single nucleotide variants and deletions in CTNNB1 ctDNA. Our study demonstrated the capability to quantitate ctDNA down to a variant allele frequency of 0.3%, achieving a sensitivity of 90% for patients at initial diagnosis, and a specificity of 100% at the patient level. Notably, ctDNA positivity correlated with tumor burden, and ctDNA levels exhibited associations with macroscopic residual disease and treatment response. Our findings provide evidence for the utility of quantitative ctDNA detection in hepatoblastoma management. Given the distinct detection targets, ctDNA and AFP-based stratification and monitoring approaches could synergize to enhance clinical decision-making. Further research is needed to elucidate the interplay between ctDNA and AFP and determine the optimal clinical applications for both methods in risk stratification and residual disease detection.

Background: Lutetium-177-prostate-specific membrane antigen- 617 (Lu-PSMA) is an effective therapy for metastatic castration-resistant prostate cancer (mCRPC). However, treatment responses are heterogeneous despite stringent positron emission tomography (PET)-based imaging selection criteria. Molecularly based biomarkers have potential to refine patient selection and optimise outcomes., Objective: To identify circulating tumour DNA (ctDNA) features associated with treatment outcomes for men treated with Lu-PSMA., Design Setting and Participants: ctDNA from men treated with Lu-PSMA in combination with idronoxil for progressive mCRPC were analysed using an 85-gene customised sequencing assay. ctDNA fractions, molecular profiles, and the presence of alterations in aggressive-variant prostate cancer (AVPC) genes were analysed at baseline, cycle 3 and at disease progression., Intervention: Men received Lu-PSMA with idronoxil every 6 wk for up to six cycles., Outcome Measurements and Statistical Analysis: Baseline and exit PSMA and fluorodeoxyglucose PET/computed tomography (CT) imaging was conducted at baseline and study exit. Single-photon emission CT (SPECT) scans were performed 24 h after Lu-PSMA. Blood samples were collected at baseline,cycle 3 and at disease progression. Cox proportional-hazards models were used to assess associations and derive hazard ratios (HRs) and confidence intervals (CIs) for associations between molecular factors, imaging features, and clinical outcomes., Results and Limitations: Sixty samples from 32 men were sequenced (32 at baseline, 24 at cycle 3, four from patients with disease progression); two samples (baseline, on-treatment) from one individual were excluded from analysis owing to poor quality of the baseline sequencing data. Alterations in AVPC genes were associated with shorter prostate-specific antigen (PSA) progression-free survival (PFS) and overall survival (OS) in univariate (HR 3.4, 95% CI 1.5-7.7; p  = 0.0036; and HR 3.3, 95% CI 1.4-7.7; p  = 0.0063, respectively) and multivariate analyses (HR 4.8, 95% CI 1.8-13; p  = 0.0014; and HR 4.1, 95% CI 1.6-11; p  = 0.004)., Conclusions: ctDNA alterations in AVPC genes were associated with shorter PSA PFS and OS among men treated with Lu-PSMA and intermittent idronoxil. These candidate molecular biomarkers warrant further study to determine whether they have predictive value and potential to guide synergistic combination strategies to enhance outcomes for men treated with Lu-PSMA for mCRPC., Patient Summary: Certain DNA/gene changes detected in the blood of men with advanced prostate cancer were associated with shorter benefit from lutetium PSMA, a targeted radioactive therapy. This information may be useful in determining which men may benefit most from this treatment, but additional research is needed., (© 2023 The Author(s).)

A Community Genetics carrier screening program for the Jewish community has operated on-site in high schools in Sydney (Australia) for 25 years. During 2020, in response to the COVID-19 pandemic, government-mandated social-distancing, 'lock-down' public health orders, and laboratory supply-chain shortages prevented the usual operation and delivery of the annual testing program. We describe development of three responses to overcome these challenges: (1) pivoting to online education sufficient to ensure informed consent for both genetic and genomic testing; (2) development of contactless telehealth with remote training and supervision for collecting genetic samples using buccal swabs; and (3) a novel patient and specimen identification 'GeneTrustee' protocol enabling fully identified clinical-grade specimens to be collected and DNA extracted by a research laboratory while maintaining full participant confidentiality and privacy. These telehealth strategies for education, consent, specimen collection and sample processing enabled uninterrupted delivery and operation of complex genetic testing and screening programs even amid pandemic restrictions. These tools remain available for future operation and can be adapted to other programs., (© 2023. The Author(s).)

The risk of Leber hereditary optic neuropathy (LHON) has largely been extrapolated from disease cohorts, which underestimate the population prevalence of pathogenic primary LHON variants as a result of incomplete disease penetrance. Understanding the true population prevalence of primary LHON variants, alongside the rate of clinical disease, provides a better understanding of disease risk and variant penetrance. We identified pathogenic primary LHON variants in whole-genome sequencing data of a well-characterized population-based control cohort and found that the prevalence is far greater than previously estimated, as it occurs in approximately 1 in 800 individuals. Accordingly, we were able to more accurately estimate population risk and disease penetrance in LHON variant carriers, validating our findings by using other large control datasets. These findings will inform accurate counseling in relation to the risk of vision loss in LHON variant carriers and disease manifestation in their family. This Matters Arising paper is in response to Lopez Sanchez et al. (2021), published in The American Journal of Human Genetics. See also the response by Mackey et al. (2022), published in this issue., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: Children on the autism spectrum are more likely to have sleep problems than non-autistic children. Sleep disturbance may exacerbate emotional and behavioral problems of children on the autism spectrum. A better understanding of the biological mechanisms underlying sleep disturbance provide clues to better management for this co-morbid condition in autism. The goal of the current study is to identify genetic variants associated with sleep disturbance and melatonin levels in autistic children., Methods: A total of 969 children on the autism spectrum were genotyped using the Global Screening Array v1 or Global Screening Array v2. Sleep problems were assessed using the Children's Sleep Habits Questionnaire (CSHQ). Melatonin levels were measured using the urine samples of 219 probands. The relationship between the melatonin level and CSHQ score was examined using the general linear model. The genetic variants associated with the CSHQ score and melatonin level as two separate quantitative traits were determined using genomewide association studies., Results: The data indicates that urine melatonin levels were positively associated with CSHQ scores, suggesting that autistic children with a poorer sleep qualiy could has higher melatonin level. Furthermore, genetic assocication studies suggest that genetic pathways involved in pro-inflammatory responses might be involved in sleep disturbance, while genetic pathways involved in catecholamine-secreting PC12 cells and Schwann cells could be associated with melatonin levels., Conclusions: Taken together, our findings indicate that sleep disturbance and melatonin metabolism could be attributable to distinct biological mechanisms in autistic children since they might not share genetic contributors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lin, Masi, Moni, Kummerfeld and Eapen.)

Background: Despite healthcare professionals (HCP) endorsing the clinical utility of pharmacogenomics testing, use in clinical practice is limited., Aims: To assess HCP' perceptions of pharmacogenomic testing and identify barriers to implementation., Methods: HCP involved in prescribing decisions at three hospitals in Sydney, Australia, were invited to participate. The online survey assessed perceptions of pharmacogenomic testing, including: (i) demographic and practice variables; (ii) use, knowledge and confidence; (iii) perceived benefits; (iv) barriers to implementation; and (v) operational and/or system changes and personnel required to implement on site., Results: HCP were predominantly medical practitioners (75/107) and pharmacists (25/107). HCP perceived pharmacogenomic testing was beneficial to identify reasons for drug intolerance (85/95) and risk of side-effects (86/95). Although testing was considered relevant to their practice (79/100), few HCP (23/100) reported past or intended future use (26/100). Few HCP reported confidence in their ability to identify indications for pharmacogenomic testing (14/107), order tests (19/106) and communicate results with patients (16/107). Lack of clinical practice guidelines (62/79) and knowledge (54/77) were identified as major barriers to implementation of pharmacogenomics. Comprehensive reimbursement for testing and clinical practice guidelines, alongside models-of-care involving multidisciplinary teams and local clinical champions were suggested as strategies to facilitate implementation of pharmacogenomic testing into practice., Conclusions: Pharmacogenomic testing was considered important to guide drug selection and dosing decisions. However, limited knowledge, low confidence and an absence of guidelines impede the use of pharmacogenomic testing. Establishment of local resources including multidisciplinary models-of-care was suggested to facilitate implementation of pharmacogenomics., (© 2022 The Authors. Internal Medicine Journal published by John Wiley & Sons Australia, Ltd on behalf of Royal Australasian College of Physicians.)

Objective: To assess the benefits and limitations of whole genome sequencing (WGS) compared to exome sequencing (ES) or multigene panel (MGP) in the molecular diagnosis of developmental and epileptic encephalopathies (DEE)., Methods: We performed WGS of 30 comprehensively phenotyped DEE patient trios that were undiagnosed after first-tier testing, including chromosomal microarray and either research ES (n = 15) or diagnostic MGP (n = 15)., Results: Eight diagnoses were made in the 15 individuals who received prior ES (53%): 3 individuals had complex structural variants; 5 had ES-detectable variants, which now had additional evidence for pathogenicity. Eleven diagnoses were made in the 15 MGP-negative individuals (68%); the majority (n = 10) involved genes not included in the panel, particularly in individuals with postneonatal onset of seizures and those with more complex presentations including movement disorders, dysmorphic features, or multiorgan involvement. A total of 42% of diagnoses were autosomal recessive or X-chromosome linked., Conclusion: WGS was able to improve diagnostic yield over ES primarily through the detection of complex structural variants (n = 3). The higher diagnostic yield was otherwise better attributed to the power of re-analysis rather than inherent advantages of the WGS platform. Additional research is required to assist in the assessment of pathogenicity of novel noncoding and complex structural variants and further improve diagnostic yield for patients with DEE and other neurogenetic disorders., (© 2021 American Academy of Neurology.)

Whole genome sequencing (WGS) has the potential to outperform clinical microarrays for the detection of structural variants (SV) including copy number variants (CNVs), but has been challenged by high false positive rates. Here we present ClinSV, a WGS based SV integration, annotation, prioritization, and visualization framework, which identified 99.8% of simulated pathogenic ClinVar CNVs > 10 kb and 11/11 pathogenic variants from matched microarrays. The false positive rate was low (1.5-4.5%) and reproducibility high (95-99%). In clinical practice, ClinSV identified reportable variants in 22 of 485 patients (4.7%) of which 35-63% were not detectable by current clinical microarray designs. ClinSV is available at https://github.com/KCCG/ClinSV .

Salivary duct carcinoma (SDCa) is a rare cancer with high rate of metastases and poor survival despite aggressive multimodality treatment. This study analyzes the genetic changes in SDCa, their impact on cancer pathways, and evaluates whether molecular patterns can identify subgroups with distinct clinical characteristics and outcome. Clinicopathologic details and tissue samples from 66 patients (48 males, 18 females) treated between 1995 and 2018 were obtained from multiple institutions. Androgen receptor (AR) was assessed by immunohistochemistry, and the Illumina TruSight 170 gene panel was used for DNA sequencing. Male gender, lympho-vascular invasion, lymph node metastasis, and smoking were significant predictors of disease-free survival. AR was present in 79%. Frequently encountered alterations were mutations in TP53 (51%), PIK3CA (32%) and HRAS (22%), as well as amplifications of CDK4/6 (22%), ERBB2 (21%), MYC (16%), and deletions of CDKN2A (13%). TP53 mutation and MYC amplifications were associated with decreased disease-free survival. Analysis of cancer pathways revealed that the PI3K pathway was most commonly affected. Alterations in the cell cycle pathway were associated with impaired disease-free survival (HR 2.6, P = 0.038). Three subgroups based on AR and ERBB2 status were identified, which featured distinct molecular patterns and outcome. Among AR positive SDCa, HRAS mutations were restricted to AR positive tumors without ERBB2 amplification and HRAS mutations featured high co-occurrence with PIK3CA alterations, which seems specific to SDCa. AR negative SDCa were associated with poor disease-free survival in multivariate analysis (HR 4.5, P = 0.010) and none of these tumors exhibited ERBB2 amplification or HRAS mutations. AR and ERBB2 status in SDCa thus classifies tumors with distinct molecular profiles relevant to future targeted therapy. Furthermore, clinical factors such as smoking and molecular features such as MYC amplification may serve as markers of poor prognosis of SDCa.

Introduction: Dystonia is a clinically and genetically heterogeneous disorder and a genetic cause is often difficult to elucidate. This is the first study to use whole genome sequencing (WGS) to investigate dystonia in a large sample of affected individuals., Methods: WGS was performed on 111 probands with heterogenous dystonia phenotypes. We performed analysis for coding and non-coding variants, copy number variants (CNVs), and structural variants (SVs). We assessed for an association between dystonia and 10 known dystonia risk variants., Results: A genetic diagnosis was obtained for 11.7% (13/111) of individuals. We found that a genetic diagnosis was more likely in those with an earlier age at onset, younger age at testing, and a combined dystonia phenotype. We identified pathogenic/likely-pathogenic variants in ADCY5 (n = 1), ATM (n = 1), GNAL (n = 2), GLB1 (n = 1), KMT2B (n = 2), PRKN (n = 2), PRRT2 (n = 1), SGCE (n = 2), and THAP1 (n = 1). CNVs were detected in 3 individuals. We found an association between the known risk variant ARSG rs11655081 and dystonia (p = 0.003)., Conclusion: A genetic diagnosis was found in 11.7% of individuals with dystonia. The diagnostic yield was higher in those with an earlier age of onset, younger age at testing, and a combined dystonia phenotype. WGS may be particularly relevant for dystonia given that it allows for the detection of CNVs, which accounted for 23% of the genetically diagnosed cases., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Gasdermin-D (GSDMD) is cleaved by caspase-1, caspase-4, and caspase-11 in response to canonical and noncanonical inflammasome activation. Upon cleavage, GSDMD oligomerizes and forms plasma membrane pores, resulting in interleukin-1β (IL-1β) secretion, pyroptotic cell death, and inflammatory pathologies, including periodic fever syndromes and septic shock-a plague on modern medicine. Here, we showed that IRF2, a member of the interferon regulatory factor (IRF) family of transcription factors, was essential for the transcriptional activation of GSDMD. A forward genetic screen with N -ethyl- N -nitrosourea (ENU)-mutagenized mice linked IRF2 to inflammasome signaling. GSDMD expression was substantially attenuated in IRF2- deficient macrophages, endothelial cells, and multiple tissues, which corresponded with reduced IL-1β secretion and inhibited pyroptosis. Mechanistically, IRF2 bound to a previously uncharacterized but unique site within the GSDMD promoter to directly drive GSDMD transcription for the execution of pyroptosis. Disruption of this single IRF2-binding site abolished signaling by both the canonical and noncanonical inflammasomes. Together, our data illuminate a key transcriptional mechanism for expression of the gene encoding GSDMD, a critical mediator of inflammatory pathologies., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Malignancies arising from mutation of tumor suppressors have unexplained tissue proclivity. For example, BAP1 encodes a widely expressed deubiquitinase for histone H2A, but germline mutations are predominantly associated with uveal melanomas and mesotheliomas. We show that BAP1 inactivation causes apoptosis in mouse embryonic stem cells, fibroblasts, liver, and pancreatic tissue but not in melanocytes and mesothelial cells. Ubiquitin ligase RNF2, which silences genes by monoubiquitinating H2A, promoted apoptosis in BAP1-deficient cells by suppressing expression of the prosurvival genes Bcl2 and Mcl1. In contrast, BAP1 loss in melanocytes had little impact on expression of prosurvival genes, instead inducing Mitf Thus, BAP1 appears to modulate gene expression by countering H2A ubiquitination, but its loss only promotes tumorigenesis in cells that do not engage an RNF2-dependent apoptotic program., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The E3 ubiquitin ligase CRL4 COP1/DET1 is active in the absence of ERK signaling, modifying the transcription factors ETV1, ETV4, ETV5, and c-JUN with polyubiquitin that targets them for proteasomal degradation. Here we show that this posttranslational regulatory mechanism is active in neurons, with ETV5 and c-JUN accumulating within minutes of ERK activation. Mice with constitutive photomorphogenesis 1 ( Cop1 ) deleted in neural stem cells showed abnormally elevated expression of ETV1, ETV4, ETV5, and c-JUN in the developing brain and spinal cord. Expression of c-JUN target genes Vimentin and Gfap was increased, whereas ETV5 and c-JUN both contributed to an expanded number of cells expressing genes associated with gliogenesis, including Olig1 , Olig2 , and Sox10. The mice had subtle morphological abnormalities in the cerebral cortex, hippocampus, and cerebellum by embryonic day 18 and died soon after birth. Elevated c-JUN, ETV5, and ETV1 contributed to the perinatal lethality, as several Cop1 -deficient mice also lacking c-Jun and Etv5 , or lacking Etv5 and heterozygous for Etv1 , were viable., Competing Interests: Conflict of interest statement: All authors were employees of Genentech., (Copyright © 2018 the Author(s). Published by PNAS.)

Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1β processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1β maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd(-/-) mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1β secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd(-/-) mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.

Paired Ig-like type 2 receptor (PILR)α inhibitory receptor and its counterpart PILRβ activating receptor are coexpressed on myeloid cells. In this article, we report that PILRα, but not PILRβ, is elevated in human rheumatoid arthritis synovial tissue and correlates with inflammatory cell infiltration. Pilrα(-/-) mice produce more pathogenic cytokines during inflammation and are prone to enhanced autoimmune arthritis. Correspondingly, engaging PILRα with anti-PILRα mAb ameliorates inflammation in mouse arthritis models and suppresses the production of proinflammatory cytokines. Our studies suggest that PILRα mediates an important inhibitory pathway that can dampen inflammatory responses., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Asthma is canonically thought of as a disorder of excessive Th2-driven inflammation in the airway, although recent studies have described heterogeneity with respect to asthma pathophysiology. We have previously described distinct phenotypes of asthma based on the presence or absence of a three-gene "Th2 signature" in bronchial epithelium, which differ in terms of eosinophilic inflammation, mucin composition, subepithelial fibrosis, and corticosteroid responsiveness. In the present analysis, we sought to describe Th2 inflammation in human asthmatic airways quantitatively with respect to known mediators of inflammation and intercellular communication. Using whole-genome microarray and quantitative real-time PCR analysis of endobronchial biopsies from 27 mild-to-moderate asthmatics and 13 healthy controls with associated clinical and demographic data, we found that asthmatic Th2 inflammation is expressed over a variable continuum, correlating significantly with local and systemic measures of allergy and eosinophilia. We evaluated a composite metric describing 79 coexpressed genes associated with Th2 inflammation against the biological space comprising cytokines, chemokines, and growth factors, identifying distinctive patterns of inflammatory mediators as well as Wnt, TGF-β, and platelet-derived growth factor family members. This integrated description of the factors regulating inflammation, cell migration, and tissue remodeling in asthmatic airways has important consequences for the pathophysiological and clinical impacts of emerging asthma therapeutics targeting Th2 inflammation.

Previous studies have suggested that the jaggedness of the healed or healing infarct edge influences cardiac electrical stability. However, these findings have been based on histological observations rather than quantitative measurements. The aim of this study was to assess infarct jaggedness by calculating its fractal dimension and to examine how this influences cardiac electrical stability during late infarct healing in the rabbit. Using programmed electrical stimulation, it was found that the fractal dimension did not differ significantly in 19 rabbits that had inducible ventricular tachycardia and 16 that did not. We conclude from these studies in the mature rabbit that infarct edge jaggedness does not influence the ease with which ventricular tachycardia is induced during late myocardial infarct healing.

This study describes the use of a shareware software package available from the National Institutes of Health for computing the fractal dimension. Specifically, when fractal analysis is used in its correct context it provides for a quantitative description of the space filling properties of two-dimensional objects. A rabbit model of post myocardial infarction is described where the cross-sectional infarct edge is reconstructed and its jaggedness determined by calculating its fractal dimension via the pixel dilation method. The fractal dimensions of the anterior and posterior lateral infarct edges were calculated to have a mean of 1.16 and 1.29, respectively. In conclusion, the fractal technique can be used to describe the complex jaggedness of the infarct edge. This case study also illustrates the fact that the complexity of an infarcted area is not uniform across the scar. For example, we found that the space filling properties of the anterior and posterior borders of a myocardial infarct can differ by more than 2-fold.