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Genetic interactions are adaptive within a species. Hybridization can disrupt such species-specific genetic interactions and creates novel interactions that alter the hybrid progeny overall fitness. Hybrid incompatibility, which refers to degenerative genetic interactions that decrease the overall hybrid survival, is one of the results from combining two diverged genomes in hybrids. The discovery of spontaneous lethal tumorigenesis and underlying genetic interactions in select hybrids between diverged Xiphophorus species showed that lethal pathological process can result from degenerative genetic interactions. Such genetic interactions leading to lethal phenotype are thought to shield gene flow between diverged species. However, hybrids between certain Xiphophorus species do not develop such tumors. Here we report the identification of a locus residing in the genome of one Xiphophorus species that represses an oncogene from a different species. Our finding provides insights into normal and pathological pigment cell development, regulation and molecular mechanism in hybrid incompatibility. Significance: The Dobzhansky-Muller model states epistatic interactions occurred between genes in diverged species underlies hybrid incompatibility. There are a few vertebrate interspecies hybrid cases that support the Dobzhansky-Muller model. This study reports a fish hybrid system where incompatible genetic interactions are involved in neuronal regulation of pigment cell biology, and also identified a novel point of regulation for pigment cells.
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BACKGROUND AND AIMS: Previously, we found that FK506 binding protein 51 (Fkbp51) knockout (KO) mice resist high fat diet-induced fatty liver and alcohol-induced liver injury. The aim of this research is to identify the mechanism of Fkbp51 in liver injury. METHODS: Carbon tetrachloride (CCl4)-induced liver injury was compared between Fkbp51 KO and wild type (WT) mice. Step-wise and in-depth analyses were applied, including liver histology, biochemistry, RNA-Seq, mitochondrial respiration, electron microscopy, and molecular assessments. The selective FKBP51 inhibitor (SAFit2) was tested as a potential treatment to ameliorate liver injury. RESULTS: Fkbp51 knockout mice exhibited protection against liver injury, as evidenced by liver histology, reduced fibrosis-associated markers and lower serum liver enzyme levels. RNA-seq identified differentially expressed genes and involved pathways, such as fibrogenesis, inflammation, mitochondria, and oxidative metabolism pathways and predicted the interaction of FKBP51, Parkin, and HSP90. Cellular studies supported co-localization of Parkin and FKBP51 in the mitochondrial network, and Parkin was shown to be expressed higher in the liver of KO mice at baseline and after liver injury relative to WT. Further functional analysis identified that KO mice exhibited increased ATP production and enhanced mitochondrial respiration. KO mice have increased mitochondrial size, increased autophagy/mitophagy and mitochondrial-derived vesicles (MDV), and reduced reactive oxygen species (ROS) production, which supports enhancement of mitochondrial quality control (MQC). Application of SAFit2, an FKBP51 inhibitor, reduced the effects of CCl4-induced liver injury and was associated with increased Parkin, pAKT, and ATP production. CONCLUSIONS: Downregulation of FKBP51 represents a promising therapeutic target for liver disease treatment.
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Patient-derived xenografts (PDX) remain valuable models for understanding the biology and for developing novel therapeutics. To expand current PDX models of childhood leukemia, we have developed new PDX models from Hispanic patients, a subgroup with a poorer overall outcome. Of 117 primary leukemia samples obtained, successful engraftment and serial passage in mice were achieved in 82 samples (70%). Hispanic patient samples engrafted at a rate (51/73, 70%) that was similar to non-Hispanic patient samples (31/45, 70%). With a new algorithm to remove mouse contamination in multi-omics datasets including methylation data, we found PDX models faithfully reflected somatic mutations, copy-number alterations, RNA expression, gene fusions, whole-genome methylation patterns, and immunophenotypes found in primary tumor (PT) samples in the first 50 reported here. This cohort of characterized PDX childhood leukemias represents a valuable resource in that germline DNA sequencing has allowed the unambiguous determination of somatic mutations in both PT and PDX.
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Subcutaneous patient-derived xenografts (PDXs) are an important tool for childhood cancer research. Here, we describe a resource of 68 early passage PDXs established from 65 pediatric solid tumor patients. Through genomic profiling of paired PDXs and patient tumors (PTs), we observe low mutational similarity in about 30% of the PT/PDX pairs. Clonal analysis in these pairs show an aggressive PT minor subclone seeds the major clone in the PDX. We show evidence that this subclone is more immunogenic and is likely suppressed by immune responses in the PT. These results suggest interplay between intratumoral heterogeneity and antitumor immunity may underlie the genetic disparity between PTs and PDXs. We further show that PDXs generally recapitulate PTs in copy number and transcriptomic profiles. Finally, we report a gene fusion LRPAP1-PDGFRA. In summary, we report a childhood cancer PDX resource and our study highlights the role of immune constraints on tumor evolution.
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Neoplasias , Animais , Criança , Humanos , Xenoenxertos , Neoplasias/genética , Neoplasias/patologia , Transcriptoma/genética , Mutação , Modelos Animais de Doenças , Genômica/métodos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Non-neuronal cells constitute 90%-95% of sensory ganglia. These cells, especially glial and immune cells, play critical roles in the modulation of sensory neurons. This study aimed to identify, profile, and summarize the types of trigeminal ganglion (TG) non-neuronal cells in naïve male mice using published and our own data generated by single-cell RNA sequencing, flow cytometry, and immunohistochemistry. TG has five types of non-neuronal cells, namely, glial, fibroblasts, smooth muscle, endothelial, and immune cells. There is an agreement among publications for glial, fibroblasts, smooth muscle, and endothelial cells. Based on gene profiles, glial cells were classified as myelinated and non-myelinated Schwann cells and satellite glial cells. Mpz has dominant expression in Schwann cells, and Fabp7 is specific for SCG. Two types of Col1a2+ fibroblasts located throughout TG were distinguished. TG smooth muscle and endothelial cells in the blood vessels were detected using well-defined markers. Our study reported three types of macrophages (Mph) and four types of neutrophils (Neu) in TG. Mph were located in the neuronal bodies and nerve fibers and were sub-grouped by unique transcriptomic profiles with Ccr2, Cx3cr1, and Iba1 as markers. A comparison of databases showed that type 1 Mph is similar to choroid plexus-low (CPlo) border-associated Mph (BAMs). Type 2 Mph has the highest prediction score with CPhi BAMs, while type 3 Mph is distinct. S100a8+ Neu were located in the dura surrounding TG and were sub-grouped by clustering and expressions of Csf3r, Ly6G, Ngp, Elane, and Mpo. Integrative analysis of published datasets indicated that Neu-1, Neu-2, and Neu-3 are similar to the brain Neu-1 group, while Neu-4 has a resemblance to the monocyte-derived cells. Overall, the generated and summarized datasets on non-neuronal TG cells showed a unique composition of myeloid cell types in TG and could provide essential and fundamental information for studies on cell plasticity, interactomic networks between neurons and non-neuronal cells, and function during a variety of pain conditions in the head and neck regions.
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Mechanisms of sex-dependent orofacial pain are widely understudied. A significant gap in knowledge exists about comprehensive regulation of tissue-specific trigeminal sensory neurons in diseased state of both sexes. Using RNA sequencing of FACS sorted retro-labeled sensory neurons innervating tongue tissue, we determined changes in transcriptomic profiles in males and female mice under naïve as well as tongue-tumor bearing conditions Our data revealed the following interesting findings: (1) FACS sorting obtained higher number of neurons from female trigeminal ganglia (TG) compared to males; (2) Naïve female neurons innervating the tongue expressed immune cell markers such as Csf1R, C1qa and others, that weren't expressed in males. This was validated by Immunohistochemistry. (3) Accordingly, immune cell markers such as Csf1 exclusively sensitized TRPV1 responses in female TG neurons. (4) Male neurons were more tightly regulated than female neurons upon tumor growth and very few differentially expressed genes (DEGs) overlapped between the sexes, (5) Male DEGs contained higher number of transcription factors whereas female DEGs contained higher number of enzymes, cytokines and chemokines. Collectively, this is the first study to characterize the effect of sex as well as of tongue-tumor on global gene expression, pathways and molecular function of tongue-innervating sensory neurons.
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Células Receptoras Sensoriais , Língua , Camundongos , Masculino , Feminino , Animais , Língua/metabolismo , Gânglio Trigeminal/metabolismo , Caracteres Sexuais , Biomarcadores/metabolismo , GenômicaRESUMO
BACKGROUND: Efficient DNA repair in response to standard chemo and radiation therapies often contributes to glioblastoma (GBM) therapy resistance. Understanding the mechanisms of therapy resistance and identifying the drugs that enhance the therapeutic efficacy of standard therapies may extend the survival of GBM patients. In this study, we investigated the role of KDM1A/LSD1 in DNA double-strand break (DSB) repair and a combination of KDM1A inhibitor and temozolomide (TMZ) in vitro and in vivo using patient-derived glioma stem cells (GSCs). METHODS: Brain bioavailability of the KDM1A inhibitor (NCD38) was established using LS-MS/MS. The effect of a combination of KDM1A knockdown or inhibition with TMZ was studied using cell viability and self-renewal assays. Mechanistic studies were conducted using CUT&Tag-seq, RNA-seq, RT-qPCR, western blot, homologous recombination (HR) and non-homologous end joining (NHEJ) reporter, immunofluorescence, and comet assays. Orthotopic murine models were used to study efficacy in vivo. RESULTS: TCGA analysis showed KDM1A is highly expressed in TMZ-treated GBM patients. Knockdown or knockout or inhibition of KDM1A enhanced TMZ efficacy in reducing the viability and self-renewal of GSCs. Pharmacokinetic studies established that NCD38 readily crosses the blood-brain barrier. CUT&Tag-seq studies showed that KDM1A is enriched at the promoters of DNA repair genes and RNA-seq studies confirmed that KDM1A inhibition reduced their expression. Knockdown or inhibition of KDM1A attenuated HR and NHEJ-mediated DNA repair capacity and enhanced TMZ-mediated DNA damage. A combination of KDM1A knockdown or inhibition and TMZ treatment significantly enhanced the survival of tumor-bearing mice. CONCLUSIONS: Our results provide evidence that KDM1A inhibition sensitizes GBM to TMZ via attenuation of DNA DSB repair pathways.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Animais , Camundongos , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Lisina/genética , Lisina/farmacologia , Lisina/uso terapêutico , Quebras de DNA de Cadeia Dupla , Espectrometria de Massas em Tandem , Linhagem Celular Tumoral , Glioma/tratamento farmacológico , Reparo do DNA , DNA/farmacologia , DNA/uso terapêutico , Histona Desmetilases/genética , Histona Desmetilases/farmacologia , Histona Desmetilases/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mechanisms of sex-dependent orofacial pain are widely understudied. A significant gap in knowledge exists about comprehensive regulation of tissue-specific trigeminal sensory neurons in diseased state of both sexes. Using RNA sequencing of FACS sorted retro-labeled sensory neurons innervating tongue tissue, we determined changes in transcriptomic profiles in males and female mice under naïve as well as tongue-tumor bearing conditions Our data revealed the following interesting findings: 1) Tongue tissue of female mice was innervated with higher number of trigeminal neurons compared to males; 2) Naïve female neurons innervating the tongue exclusively expressed immune cell markers such as Csf1R, C1qa and others, that weren't expressed in males. This was validated by Immunohistochemistry. 4) Accordingly, immune cell markers such as Csf1 exclusively sensitized TRPV1 responses in female TG neurons. 3) Male neurons were more tightly regulated than female neurons upon tumor growth and very few differentially expressed genes (DEGs) overlapped between the sexes, 5) Male DEGs contained higher number of transcription factors whereas female DEGs contained higher number of enzymes, cytokines and chemokines. Collectively, this is the first study to characterize the effect of sex as well as of tongue-tumor on global gene expression, pathways and molecular function of tongue-innervating sensory neurons.
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Pain development and resolution patterns in many diseases are sex-dependent. This study aimed to develop pain models with sex-dependent resolution trajectories, and identify factors linked to resolution of pain in females and males. Using different intra-plantar (i.pl.) treatment protocols with prolactin (PRL), we established models with distinct, sex-dependent patterns for development and resolution of pain. An acute PRL-evoked pain trajectory, in which hypersensitivity is fully resolved within 1 day, showed substantial transcriptional changes after pain-resolution in female and male hindpaws and in the dorsal root ganglia (DRG). This finding supports the notion that pain resolution is an active process. Prolonged treatment with PRL high dose (1 µg) evoked mechanical hypersensitivity that resolved within 5-7 days in mice of both sexes and exhibited a pro-inflammatory transcriptional response in the hindpaw, but not DRG, at the time point preceding resolution. Flow cytometry analysis linked pro-inflammatory responses in female hindpaws to macrophages/monocytes, especially CD11b+/CD64+/MHCII+ cell accumulation. Prolonged low dose PRL (0.1 µg) treatment caused non-resolving mechanical hypersensitivity only in females. This effect was independent of sensory neuronal PRLR and was associated with a lack of immune response in the hindpaw, although many genes underlying tissue damage were affected. We conclude that different i.pl. PRL treatment protocols generates distinct, sex-specific pain hypersensitivity resolution patterns. PRL-induced pain resolution is preceded by a pro-inflammatory macrophage/monocyte-associated response in the hindpaws of mice of both sexes. On the other hand, the absence of a peripheral inflammatory response creates a permissive condition for PRL-induced pain persistency in females.
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Prolactina , Receptores da Prolactina , Animais , Feminino , Gânglios Espinais , Masculino , Camundongos , Dor , Prolactina/farmacologia , Receptores da Prolactina/genética , Células Receptoras SensoriaisRESUMO
Obesity is an enormous global health problem, and obesity-induced nonalcoholic steatohepatitis (NASH) is contributing to a rising incidence and mortality for hepatocellular carcinoma (HCC). Increase in de novo lipogenesis and decrease in fatty acid ß-oxidation (FAO) underlie hepatic lipid accumulation in NASH. Astrocyte-elevated gene-1/metadherin (AEG-1) overexpression contributes to both NASH and HCC. AEG-1 harbors an LXXLL motif through which it blocks activation of peroxisome proliferator activated receptor α (PPARα), a key regulator of FAO. To better understand the role of LXXLL motif in mediating AEG-1 function, using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, we generated a mouse model (AEG-1-L24K/L25H) in which the LXXLL motif in AEG-1 was mutated to LXXKH. We observed increased activation of PPARα in AEG-1-L24K/L25H livers providing partial protection from high-fat diet-induced steatosis. Interestingly, even with equal gene dosage levels, compared with AEG-1-wild-type livers, AEG-1-L24K/L25H livers exhibited increase in levels of lipogenic enzymes, mitogenic activity and inflammation, which are attributes observed when AEG-1 is overexpressed. These findings indicate that while LXXLL motif favors steatotic activity of AEG-1, it keeps in check inflammatory and oncogenic functions, thus maintaining a homeostasis in AEG-1 function. AEG-1 is being increasingly appreciated as a viable target for ameliorating NASH and NASH-HCC, and as such, in-depth understanding of the functions and molecular attributes of this molecule is essential. Conclusion: The present study unravels the unique role of the LXXLL motif in mediating the balance between the metabolic and oncogenic functions of AEG-1.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteínas de Membrana , Hepatopatia Gordurosa não Alcoólica , Proteínas de Ligação a RNA , Animais , Astrócitos/metabolismo , Carcinogênese/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteínas de Membrana/genética , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Obesidade/genética , PPAR alfa/genética , Proteínas de Ligação a RNA/genética , Fatores de TranscriçãoRESUMO
Trigeminal (TG), dorsal root (DRG), and nodose/jugular (NG/JG) ganglia each possess specialized and distinct functions. We used RNA sequencing of two-cycle sorted Pirt-positive neurons to identify genes exclusively expressing in L3-L5 DRG, T10-L1 DRG, NG/JG, and TG mouse ganglion neurons. Transcription factor Phox2b and Efcab6 are specifically expressed in NG/JG while Hoxa7 is exclusively present in both T10-L1 and L3-L5 DRG neurons. Cyp2f2, Krt18, and Ptgds, along with pituitary hormone prolactin (Prl), growth hormone (Gh), and proopiomelanocortin (Pomc) encoding genes are almost exclusively in TG neurons. Immunohistochemistry confirmed selective expression of these hormones in TG neurons and dural nerves; and showed GH expression in subsets of TRPV1+ and CGRP+ TG neurons. We next examined GH roles in hypersensitivity in the spinal versus trigeminal systems. Exogenous GH produced mechanical hypersensitivity when injected intrathecally, but not intraplantarly. GH-induced thermal hypersensitivity was not detected in the spinal system. GH dose-dependently generated orofacial and headache-like periorbital mechanical hypersensitivity after administration into masseter muscle and dura, respectively. Periorbital mechanical hypersensitivity was reversed by a GH receptor antagonist, pegvisomant. Overall, pituitary hormone genes are selective for TG versus other ganglia somatotypes; and GH has distinctive functional significance in the trigeminal versus spinal systems.
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Hormônio do Crescimento/metabolismo , Dor/metabolismo , Pró-Opiomelanocortina/metabolismo , Prolactina/metabolismo , Células Receptoras Sensoriais/metabolismo , Gânglio Trigeminal/metabolismo , Animais , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Camundongos , Camundongos Transgênicos , Gânglio Nodoso/citologia , Gânglio Nodoso/metabolismo , Gânglio Trigeminal/citologiaRESUMO
Chlamydia trachomatis is an obligate intracellular bacterium whose unique developmental cycle consists of an infectious elementary body and a replicative reticulate body. Progression of this developmental cycle requires temporal control of the transcriptome. In addition to the three chlamydial sigma factors (σ66, σ28, and σ54) that recognize promoter sequences of genes, chlamydial transcription factors are expected to play crucial roles in transcriptional regulation. Here, we investigate the function of GrgA, a Chlamydia-specific transcription factor, in C. trachomatis transcriptomic expression. We show that 10 to 30 min of GrgA overexpression induces 13 genes, which likely comprise the direct regulon of GrgA. Significantly, σ66-dependent genes that code for two important transcription repressors are components of the direct regulon. One of these repressors is Euo, which prevents the expression of late genes during early phases. The other is HrcA, which regulates molecular chaperone expression and controls stress response. The direct regulon also includes a σ28-dependent gene that codes for the putative virulence factor PmpI. Furthermore, overexpression of GrgA leads to decreased expression of almost all tRNAs. Transcriptomic studies suggest that GrgA, Euo, and HrcA have distinct but overlapping indirect regulons. These findings, together with temporal expression patterns of grgA, euo, and hrcA, indicate that a transcriptional regulatory network of these three transcription factors plays critical roles in C. trachomatis growth and development. IMPORTANCE Chlamydia trachomatis is the most prevalent sexually transmitted bacterial pathogen worldwide and is a leading cause of preventable blindness in underdeveloped areas as well as some developed countries. Chlamydia carries genes that encode a limited number of known transcription factors. While Euo is thought to be critical for early chlamydial development, the functions of GrgA and HrcA in the developmental cycle are unclear. Activation of euo and hrcA immediately following GrgA overexpression indicates that GrgA functions as a master transcriptional regulator. In addition, by broadly inhibiting tRNA expression, GrgA serves as a key regulator of chlamydial protein synthesis. Furthermore, by upregulating pmpI, GrgA may act as an upstream virulence determinant. Finally, genes coregulated by GrgA, Euo, and HrcA likely play critical roles in chlamydial growth and developmental control.
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BACKGROUND: Studies of the gut microbiome are becoming increasingly important. Such studies require stool collections that can be processed or frozen in a timely manner so as not to alter the microbial content. Due to the logistical difficulties of home-based stool collection, there has been a challenge in selecting the appropriate sample collection technique and comparing results from different microbiome studies. Thus, we compared stool collection and two alternative clinic-based fecal microbiome collection techniques, including a newer glove-based collection method. RESULTS: We prospectively enrolled 22 adult men from our prostate cancer screening cohort SABOR (San Antonio Biomarkers of Risk for prostate cancer) in San Antonio, TX, from 8/2018 to 4/2019. A rectal swab and glove tip sample were collected from each participant during a one-time visit to our clinics. A single stool sample was collected at the participant's home. DNA was isolated from the fecal material and 16 s rRNA sequencing of the V1-V2 and V3-V4 regions was performed. We found the gut microbiome to be similar in richness and evenness, noting no differences in alpha diversity among the collection methods. The stool collection method, which remains the gold-standard method for the gut microbiome, proved to have different community composition compared to swab and glove tip techniques (p< 0.001) as measured by Bray-Curtis and unifrac distances. There were no significant differences in between the swab and glove tip samples with regard to beta diversity (p> 0.05). Despite differences between home-based stool and office-based fecal collection methods, we noted that the distance metrics for the three methods cluster by participant indicating within-person similarities. Additionally, no taxa differed among the methods in a Linear Discriminant Analysis Effect Size (LEfSe) analysis comparing all-against-all sampling methods. CONCLUSION: The glove tip method provides similar gut microbiome results as rectal swab and stool microbiome collection techniques. The addition of a new office-based collection technique could help easy and practical implementation of gut microbiome research studies and clinical practice.
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Bactérias/classificação , Fezes/microbiologia , Luvas Cirúrgicas/microbiologia , RNA Ribossômico 16S/genética , Reto/microbiologia , Manejo de Espécimes/instrumentação , Idoso , Idoso de 80 Anos ou mais , Bactérias/genética , Bactérias/isolamento & purificação , DNA Bacteriano/genética , DNA Ribossômico/genética , Microbioma Gastrointestinal , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Filogenia , Estudos Prospectivos , Análise de Sequência de DNA/métodos , Manejo de Espécimes/métodosRESUMO
Cells reprogram their transcriptome in response to stress, such as heat shock. In free-living bacteria, the transcriptomic reprogramming is mediated by increased DNA-binding activity of heat shock sigma factors and activation of genes normally repressed by heat-induced transcription factors. In this study, we performed transcriptomic analyses to investigate heat shock response in the obligate intracellular bacterium Chlamydia trachomatis, whose genome encodes only three sigma factors and a single heat-induced transcription factor. Nearly one-third of C. trachomatis genes showed statistically significant (≥1.5-fold) expression changes 30 min after shifting from 37 to 45°C. Notably, chromosomal genes encoding chaperones, energy metabolism enzymes, type III secretion proteins, as well as most plasmid-encoded genes, were differentially upregulated. In contrast, genes with functions in protein synthesis were disproportionately downregulated. These findings suggest that facilitating protein folding, increasing energy production, manipulating host activities, upregulating plasmid-encoded gene expression, and decreasing general protein synthesis helps facilitate C. trachomatis survival under stress. In addition to relieving negative regulation by the heat-inducible transcriptional repressor HrcA, heat shock upregulated the chlamydial primary sigma factor σ66 and an alternative sigma factor σ28. Interestingly, we show for the first time that heat shock downregulates the other alternative sigma factor σ54 in a bacterium. Downregulation of σ54 was accompanied by increased expression of the σ54 RNA polymerase activator AtoC, thus suggesting a unique regulatory mechanism for reestablishing normal expression of select σ54 target genes. Taken together, our findings reveal that C. trachomatis utilizes multiple novel survival strategies to cope with environmental stress and even to replicate. Future strategies that can specifically target and disrupt Chlamydia's heat shock response will likely be of therapeutic value.
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Innate lymphoid cells (ILCs) are a heterogeneous family of immune regulators that protect against mucosal pathogens but can also promote intestinal pathology. Although the plasticity between ILCs populations has been described, the role of mucosal pathogens in inducing ILC conversion leading to intestinal pathology remains unclear. Here we demonstrate that IFNγ-producing ILCs are responsible for promoting intestinal pathology in a mouse model of enterocolitis caused by Campylobacter jejuni, a common human enteric pathogen. Phenotypic analysis revealed a distinct population of IFNγ-producing NK1.1-T-bet+ILCs that accumulated in the intestine of C. jejuni-infected mice. Adoptive transfer experiments demonstrated their capacity to promote intestinal pathology. Inactivation of T-bet in NKp46+ ILCs ameliorated disease. Transcriptome analysis and cell-fate mapping experiments revealed that IFNγ-producing NK1.1-ILCs correspond to ILC1 profile and develop from RORγt+ progenitors. Collectively, we identified a distinct population of NK1.1-ex-ILC3s that promotes intestinal pathology through IFNγ production in response to C. jejuni infection.
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Infecções por Campylobacter/imunologia , Campylobacter jejuni/fisiologia , Colite/imunologia , Intestinos/imunologia , Linfócitos/imunologia , Animais , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Humanos , Imunidade Inata , Interferon gama/metabolismo , Interleucina-10/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Células Th1/imunologiaRESUMO
INTRODUCTION: Comprehensive mRNA sequencing is a powerful tool for conducting unbiased, quantitative differential gene expression analysis. However, the reliability of these data is contingent on the extraction of high-quality RNA from samples. Preserving RNA integrity during extraction can be problematic, especially in tissues such as skin with dense, connective matrices and elevated ribonuclease expression. This is a major barrier to understanding the influences of altered gene expression in many preclinical pain models and clinical pain disorders where skin is the site of tissue injury. OBJECTIVE: This study developed and evaluated extraction protocols for skin and other tissues to maximize recovery of high-integrity RNA needed for quantitative mRNA sequencing. METHODS: Rodent and human tissue samples underwent one of the several different protocols that combined either RNA-stabilizing solution or snap-freezing with bead milling or cryosectioning. Indices of RNA integrity and purity were assessed for all samples. RESULTS: Extraction of high-integrity RNA is highly dependent on the methods used. Bead-milling skin collected in RNA-stabilizing solution resulted in extensive RNA degradation. Snap-freezing in liquid nitrogen was required for skin and highly preferable for other tissues. Skin also required cryosectioning to achieve effective penetration of RNA-stabilizing solution to preserve RNA integrity, whereas bead milling could be used instead with other tissues. Each method was reproducible across multiple experimenters. Electrophoretic anomalies that skewed RNA integrity value assignment required manual correction and often resulted in score reduction. CONCLUSION: To achieve the potential of quantitative differential gene expression analysis requires verification of tissue-dependent extraction methods that yield high-integrity RNA.
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Genotyping of a 615 kb region within 8q24 with 49 haplotype-tagged single-nucleotide polymorphisms (SNPs) in 2109 samples (797 cases and 1312 controls) of two ethnic/racial groups found SNPs that are significantly associated with the risk for prostate cancer (PCa). The highest significance in Caucasian men was found for rs6983267; the AA genotype reduced the risk for PCa [odds ratio (OR) = 0.48, 95% confidence interval (CI) = 0.35-0.65, P = 2.74 x 10(-6)]. This SNP also had a significant independent effect from other SNPs in the region in this group. In Hispanic men, rs7837328 and rs921146 showed independent effects (OR = 2.55, 95% CI = 1.51-4.31, P = 4.33 x 10(-4), OR = 2.09, 95% CI = 1.40-3.12, P = 3.13 x 10(-4), respectively). Significant synergist effects for increasing numbers of high-risk alleles were found in both ethnicities. Haplotype analysis revealed major haplotypes, containing the non-risk alleles, conferred protection against PCa. We found high linkage disequilibrium between significant SNPs within the region and SNPs within the CUB and Sushi Multiple Domains 1 gene (CSMD1), on the short arm of chromosome 8 in both ethnicities. These data suggest that multiple interacting SNPs within 8q24, as well as different regions on chromosome 8 far beyond this 8q24 candidate region, may confer increased risk of PCa. This is the first report to investigate the involvement of 8q24 variants in the susceptibility for PCa in Hispanic men.
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Cromossomos Humanos Par 8/genética , Haplótipos/genética , Hispânico ou Latino/genética , Polimorfismo de Nucleotídeo Único/genética , Neoplasias da Próstata/genética , População Branca/genética , Idoso , Estudos de Casos e Controles , Estudos de Coortes , DNA de Neoplasias/genética , Variação Genética , Genótipo , Humanos , Desequilíbrio de Ligação , Escore Lod , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Estudos Prospectivos , Neoplasias da Próstata/patologia , Medição de RiscoRESUMO
To estimate the prostate cancer risk conferred by individual single nucleotide polymorphisms (SNPs), SNP-SNP interactions, and/or cumulative SNP effects, we evaluated the association between prostate cancer risk and the genetic variants of 12 key genes within the steroid hormone pathway (CYP17, HSD17B3, ESR1, SRD5A2, HSD3B1, HSD3B2, CYP19, CYP1A1, CYP1B1, CYP3A4, CYP27B1, and CYP24A1). A total of 116 tagged SNPs covering the group of genes were analyzed in 2,452 samples (886 cases and 1,566 controls) in three ethnic/racial groups. Several SNPs within CYP19 were significantly associated with prostate cancer in all three ethnicities (P = 0.001-0.009). Genetic variants within HSD3B2 and CYP24A1 conferred increased risk of prostate cancer in non-Hispanic or Hispanic Caucasians. A significant gene-dosage effect for increasing numbers of potential high-risk genotypes was found in non-Hispanic and Hispanic Caucasians. Higher-order interactions showed a seven-SNP interaction involving HSD17B3, CYP19, and CYP24A1 in Hispanic Caucasians (P = 0.001). In African Americans, a 10-locus model, with SNPs located within SRD5A2, HSD17B3, CYP17, CYP27B1, CYP19, and CYP24A1, showed a significant interaction (P = 0.014). In non-Hispanic Caucasians, an interaction of four SNPs in HSD3B2, HSD17B3, and CYP19 was found (P < 0.001). These data are consistent with a polygenic model of prostate cancer, indicating that multiple interacting genes of the steroid hormone pathway confer increased risk of prostate cancer.
Assuntos
Predisposição Genética para Doença , Hormônios Esteroides Gonadais/genética , Hormônios Esteroides Gonadais/metabolismo , Neoplasias da Próstata/genética , Idoso , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Fatores de RiscoRESUMO
UNLABELLED: Mutations in Sequestosome 1 (SQSTM1) have been shown to segregate with familial Paget's disease of bone (PDB). We examined the coding sequence of SQSTM1 in five PDB pedigrees and found three novel mutations clustered around the C-terminal ubiquitin associated domain. Disruptions of the C-terminal domain of SQSTM1 seem to be a leading cause of familial PDB. INTRODUCTION: The characteristic features of Paget's disease of bone (PDB) are caused by focal areas of excessive and uncoordinated bone remodeling. A total of seven genetic loci (PDB1-PDB7) have been reported to be associated with the disease. The gene for Sequestosome 1 (p62; SQSTM1) has been identified as the causative gene for PDB3 in numerous French-Canadian families and families predominantly of British descent. To date, a total of three mutations, all affecting the ubiquitin-associated domain of SQSTM1, have been identified: a single 1215 C to T (P392L) transversion in exon 8, a T insertion in exon 8 (E396X), and a G to A mutation at the splice junction of exon 7 (IVS7 + 1). MATERIALS AND METHODS: DNA was isolated from blood collected from the members of five U.S. PDB pedigrees. Mutation analysis of the coding sequence of the SQSTM1 gene was performed on the proband and other key individuals in the pedigrees. RESULTS: Four of the five families had SQSTM1 mutations. Three of these mutations were novel: a single base deletion in exon 8 at position 1210 (1210delT) resulting in a premature stop codon at amino acid 394, a single C deletion in exon 8 at position 1215 (1215delC) also resulting in a premature stop codon at amino acid 394, and a single 1200 C to T (P387L) transversion in exon 7. CONCLUSION: Noteworthy is the fact that these three SQSTM1 mutations, in addition to the three previously described mutations, are clustered near the C-terminal of the protein. These mutations may be acting in a dominant-negative fashion to disrupt the ubiquitin-binding function, which could result in abnormal activation of the NF-kappaB pathway and the subsequent activation of the osteoclasts. These findings imply that SQSTM1 mutations may play a role in the majority of familial PDB in the United States.