Whole-genome Profiling Research Articles

Comparative transcriptome, physiological and biochemical analyses reveal response mechanism mediated by CBF4 and ICE2 in enhancing cold stress tolerance in Gossypium thurberi.

Low temperature is one of the key environmental stresses that impair plant growth and significantly restricts the productivity and spatial distribution of crop plants. Gossypium thurberi, a wild diploid cotton species, has adapted to a wide range of temperatures and exhibits a better tolerance to chilling stress. Here, we compared phenotypes and physiochemical changes in G. thurberi under cold stress and found this species indeed showed better cold tolerance. Therefore, to understand the molecular mechanisms of the cold tolerance in G. thurberi, we compared transcription changes in leaves of G. thurberi under cold stress by high-throughput transcriptome sequencing. In total, 35 617 unigenes were identified in the whole-genome transcription profile, and 4226 differentially expressed genes (DEGs) were discovered in the leaves upon cold treatment. Gene Ontology (GO) classification analyses showed that the majority of DEGs belonged to categories of signal transduction, transcription factors (TFs) and carbohydrate transport and metabolism. The expression of several cold-responsive genes such as ICE1, CBF4, RAP2-7 and abscisic acid (ABA) biosynthesis genes involved in different signalling pathways were induced after G. thurberi seedlings were exposed to cold stress. Furthermore, cold sensitivity was increased in CBF4 and ICE2 virus-induced gene silencing (VIGS) plants, and high level of malondialdehyde (MDA) showed that the CBF4 and ICE2 silenced plants were under oxidative stress compared to their wild types, which relatively had higher levels of antioxidant enzyme activity, as evident by high levels of proline and superoxide dismutase (SOD) content. In conclusion, our findings reveal a new regulatory network of cold stress response in G. thurberi and broaden our understanding of the cold tolerance mechanism in cotton, which might accelerate functional genomics studies and genetic improvement for cold stress tolerance in cultivated cotton.

Abstract 446: Plasma genome sequencing identifies prostate cancer patients that are sensitive to platinum-based therapy

Abstract Castration-resistant prostate cancer (CRPC) is a lethal disease. A subset of these patients present with atypical clinical characteristics and aggressive disease behavior. These patients, classified as Aggressive Variant Prostate Cancer (AVPC), may derive benefit from platinum-based chemotherapy. However, the identification of AVPC patients is often challenging due to heterogeneity in clinical presentations and potentially in biological drivers, which may not be fully reflected in a biopsy obtained from a single tumor site. To address these challenges, we developed an unbiased genome sequencing method called PEGASUS (Plasma Exome and Genome Analysis by Size-Selection and Unbiased Sequencing) that enables the simultaneous detection of copy number aberrations and exome mutations from circulating-tumor DNA (ctDNA). We applied this method to plasma specimens obtained from 160 CRPC patients with and without AVPC participating in a randomized trial of cabazitaxel alone or in combination with carboplatin. We were able to successfully isolate ctDNA (>2 ng) for genomic profiling using PEGASUS in 91/160 (57%) CRPC patients. Among these 91 CRPC patients, we detected common prostate cancer mutations as well as genomic copy number changes in genes such as RB1, PTEN and TP53 and several other genes associated with DNA repair. One of the most salient features that distinguished the patients’ outcomes was whether they had diploid or aneuploid genomes detected by whole genome profiling of the ctDNA. Patients with aneuploid ctDNA had worse progression-free and overall survival. Overall, our data shows the feasibility of performing whole-genome and exome profiling of ctDNA in CRPC patients to characterize the molecular features associated with distinct clinical presentations. Candidate markers associated to benefit from platinum-based chemotherapy are being evaluated. These results pave the way for future clinical applications in biomarker discovery to assist treatment decisions in prostate cancer patients. Citation Format: Naveen Ramesh, Emi Sei, Pei Ching Tsai, Christopher Logothetis, Paul Corn, Ana Aparicio, Amado J. Zurita, Nicholas E. Navin. Plasma genome sequencing identifies prostate cancer patients that are sensitive to platinum-based therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 446.

Abstract 474: Genome-wide DNA methylation profiling reveals novel candidate epigenetic gatekeepers in hepatocarcinogenesis

Abstract Epigenetic deregulation is a critical event in human malignancies. Besides mutations in TERT promoter, found in 20% of dysplastic nodules, little is known about the key molecular alterations driving early hepatocarcinogenesis. The aims of this study were: 1) to analyze DNA methylation changes during the transition from preneoplastic lesions to early hepatocellular carcinoma (HCC), and 2) to identify candidate epigenetic gatekeepers in the transition between dysplasia and early tumors. Methylome profiling was done with Illumina HumanMethylation450 (485,000 CpG, 96% of known CpG islands). We evaluated differentially methylated CpG sites between groups using an F-test. To detect novel epigenetic gatekeepers we defined hypermethylation as a B value higher than 0.5. We profiled 144 fresh-frozen tissues from resection or liver transplant specimens and integrated these data with our previously report on DNA methylation in HCC (Villanueva et al. Hepatology 2015) resulting in: 16 normal liver, 139 cirrhosis, 8 dysplastic nodules and 227 HCC samples. Correlation between methylation and RNA expression (n= 361) was quantified with the Pearson’s coefficient. Patients were mostly male (76%), with a median age of 66, and with underlying liver disease mainly due to hepatitis C (43%) and B (23%). All tumors were early stage according to BCLC classification, including 43 cases below 2 cm in diameter. A phylo-epigenetic tree derived from the euclidean distances between differentially DNA methylated sites (n=421,997) reveals a gradient of methylation changes that spans normal, cirrhotic, dysplastic nodules and HCC. Epigenetic analysis confirmed closer proximity of dysplasia to HCC than to cirrhotic tissue. We validated aberrant methylation of previously reported HCC epidrivers within the unpublished dataset (e.g. EFBN2, TBX15) (Villanueva et al. Hepatology 2015). Focusing on CpG sites located in promoter regions (i.e., TSS200, TSS1500, 5’UTR, and 1st exon), we selected candidates hypermethylated in less than 1% of normal and cirrhotic tissue, in all high grade dysplastic nodules, and in more than 50% of small HCC nodules (<2 cm). When we integrated DNA methylation and gene expression we found a significant (all P<0.001) inverse correlation in TSPYL5 (r=-0.31), KCNA3 (r=-0.33), LDBH (r=-0.46) and SPINT2 (r=-0.43). Whole-genome DNA methylation profiles accurately discriminate between different histological lesions along the human hepatocarcinogenesis spectrum. We report novel epigenetic gatekeepers in the transition between dysplastic nodules and early HCC. Citation Format: Gabriela Hernandez Meza, Johann von Felden, Amanda Craig, Sergi Sayols, Anna Portela, Manel Esteller, Myron Schwartz, Vincenzo Mazzaferro, Josep Llovet, Augusto Villanueva. Genome-wide DNA methylation profiling reveals novel candidate epigenetic gatekeepers in hepatocarcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 474.

A Study of the Association between Breastfeeding and DNA Methylation in Peripheral Blood Cells of Infants

This article reports on new data on the association of breastfeeding with DNA methylation in the peripheral blood cells of 37 children aged from 9 months to four years. Whole-genome DNA methylation profiling was performed using the Illumina Methylation EPIC array. The Epigenome-Wide Association Study (EWAS) revealed an association between the duration of breastfeeding and the methylation level of 4276 СpG sites related to 2635 genes. According to the functional annotation, these genes were predominantly involved in the control of cell signaling systems, the development of anatomical structures and cells, and, above all, were related to the development and function of the immune system and the CNS. The results of the study allowed assuming a special role of the oxytocin signaling pathway, as a potential trigger of coordinated epigenetic changes in the genes involved in the CNS function in response to breastfeeding.

Ceftazidime-Avibactam in Combination With Fosfomycin: A Novel Therapeutic Strategy Against Multidrug-Resistant Pseudomonas aeruginosa.

Previously, by targeting penicillin-binding protein 3, Pseudomonas-derived cephalosporinase (PDC), and MurA with ceftazidime-avibactam-fosfomycin, antimicrobial susceptibility was restored among multidrug-resistant (MDR) Pseudomonas aeruginosa. Herein, ceftazidime-avibactam-fosfomycin combination therapy against MDR P. aeruginosa clinical isolate CL232 was further evaluated. Checkerboard susceptibility analysis revealed synergy between ceftazidime-avibactam and fosfomycin. Accordingly, the resistance elements present and expressed in P. aeruginosa were analyzed using whole-genome sequencing and transcriptome profiling. Mutations in genes that are known to contribute to β-lactam resistance were identified. Moreover, expression of blaPDC, the mexAB-oprM efflux pump, and murA were upregulated. When fosfomycin was administered alone, the frequency of mutations conferring resistance was high; however, coadministration of fosfomycin with ceftazidime-avibactam yielded a lower frequency of resistance mutations. In a murine infection model using a high bacterial burden, ceftazidime-avibactam-fosfomycin significantly reduced the P. aeruginosa colony-forming units (CFUs), by approximately 2 and 5 logs, compared with stasis and in the vehicle-treated control, respectively. Administration of ceftazidime-avibactam and fosfomycin separately significantly increased CFUs, by approximately 3 logs and 1 log, respectively, compared with the number at stasis, and only reduced CFUs by approximately 1 log and 2 logs, respectively, compared with the number in the vehicle-treated control. Thus, the combination of ceftazidime-avibactam-fosfomycin was superior to either drug alone. By employing a "mechanism-based approach" to combination chemotherapy, we show that ceftazidime-avibactam-fosfomycin has the potential to offer infected patients with high bacterial burdens a therapeutic hope against infection with MDR P. aeruginosa that lack metallo-β-lactamases.

5-Hydroxymethylcytosine Profiles Are Prognostic of Outcome in Neuroblastoma and Reveal Transcriptional Networks That Correlate With Tumor Phenotype.

PURPOSEWhole-genome profiles of the epigenetic modification 5-hydroxymethylcytosine (5-hmC) are robust diagnostic biomarkers in adult patients with cancer. We investigated if 5-hmC profiles would serve as novel prognostic markers in neuroblastoma, a clinically heterogeneous pediatric cancer. Because this DNA modification facilitates active gene expression, we hypothesized that 5-hmC profiles would identify transcriptomic networks driving the clinical behavior of neuroblastoma.PATIENTS AND METHODSNano-hmC-Seal sequencing was performed on DNA from Discovery (n = 51), Validation (n = 38), and Children’s Oncology Group (n = 20) cohorts of neuroblastoma tumors. RNA was isolated from 48 tumors for RNA sequencing. Genes with differential 5-hmC or expression between clusters were identified using DESeq2. A 5-hmC model predicting outcome in high-risk patients was established using linear discriminant analysis.RESULTSComparison of low- versus high-risk tumors in the Discovery cohort revealed 577 genes with differential 5-hmC. Hierarchical clustering of tumors from the Discovery and Validation cohorts using these genes identified two main clusters highly associated with established prognostic markers, clinical risk group, and outcome. Genes with increased 5-hmC and expression in the favorable cluster were enriched for pathways of neuronal differentiation and KRAS activation, whereas genes involved in inflammation and the PRC2 complex were identified in the unfavorable cluster. The linear discriminant analysis model trained on high-risk Discovery cohort tumors was prognostic of outcome when applied to high-risk tumors from the Validation and Children’s Oncology Group cohorts (hazard ratio, 3.8).CONCLUSION5-hmC profiles may be optimal DNA-based biomarkers in neuroblastoma. Analysis of transcriptional networks regulated by these epigenomic modifications may lead to a deeper understanding of drivers of neuroblastoma phenotype.

De novo DNA methylation programs regulate T cell exhaustion and limit T cell-based immunotherapies

Abstract T cell-based immunotherapies have emerged as one of the most promising frontiers in the fight against cancer and chronic infections. However, it has become clear that prolonged exposure of T cells to their cognate antigen drives them toward a terminally differentiated state, limiting their capacity to mount an effector response. The commitment of T cells to this “exhausted” fate is currently a major barrier in the advancement of T cell immunotherapy efforts. To better understand mechanisms that reinforce the T cell exhaustion gene expression program, we investigated the role of de novo epigenetic programming in establishing exhausted T cells that are non-responsive during immune checkpoint blockade (ICB) therapy. Using mouse models of tumor and chronic viral infection, we observed that genetic deletion of the de novo DNA methyltransferase, Dnmt3a, in antigen-specific T cells that are chronically stimulated allowed them to remain highly functional despite expressing high levels of the checkpoint inhibitory receptor PD-1. Furthermore, PD-1 blockade treatment resulted in massive expansion of PD-1+ Dnmt3a-deficient antigen-specific T cells. Building upon the findings from our murine studies, we have generated whole-genome DNA methylation profiles of human CD8 T cells from a wide array of differentiation states, and established an epigenetic-based human T cell multipotency index. Using this index, we report that tumor-associated PD-1hi CD8 T cells from pediatric solid tumors acquire an epigenetically reinforced terminal differentiation program. Collectively, these data establish Dnmt3a-mediated de novo DNA methylation programming as a key factor in limiting CD8 T cell-based immunotherapuetic approaches.

Stem Cell Proliferation Is Kept in Check by the Chromatin Regulators Kismet/CHD7/CHD8 and Trr/MLL3/4

SummaryChromatin remodeling accompanies differentiation, however, its role in self-renewal is less well understood. We report that in Drosophila, the chromatin remodeler Kismet/CHD7/CHD8 limits intestinal stem cell (ISC) number and proliferation without affecting differentiation. Stem-cell-specific whole-genome profiling of Kismet revealed its enrichment at transcriptionally active regions bound by RNA polymerase II and Brahma, its recruitment to the transcription start site of activated genes and developmental enhancers and its depletion from regions bound by Polycomb, Histone H1, and heterochromatin Protein 1. We demonstrate that the Trithorax-related/MLL3/4 chromatin modifier regulates ISC proliferation, colocalizes extensively with Kismet throughout the ISC genome, and co-regulates genes in ISCs, including Cbl, a negative regulator of Epidermal Growth Factor Receptor (EGFR). Loss of kismet or trr leads to elevated levels of EGFR protein and signaling, thereby promoting ISC self-renewal. We propose that Kismet with Trr establishes a chromatin state that limits EGFR proliferative signaling, preventing tumor-like stem cell overgrowths.

Whole-Genome Promoter Profiling of Plasma DNA Exhibits Diagnostic Value for Placenta-Origin Pregnancy Complications

Placenta-origin pregnancy complications, including preeclampsia (PE), gestational diabetes mellitus (GDM), fetal growth restriction (FGR), and macrosomia (MA) are common occurrences in pregnancy, resulting in significant morbidity and mortality for both mother and fetus. However, despite their frequency, there are no reliable methods for the early diagnosis of these complications. Since cfDNA is mainly derived from placental trophoblasts and maternal hematopoietic cells, it might have information for gene expression which can be used for disease prediction. Here, low coverage whole-genome sequencing on plasma DNA from 2,199 pregnancies is performed based on retrospective cohorts of 3,200 pregnant women. Read depth in the promoter regions is examined to define read-depth distribution patterns of promoters for pregnancy complications and controls. Using machine learning methods, classifiers for predicting pregnancy complications are developed. Using these classifiers, complications are successfully predicted with an accuracy of 80.3%, 78.9%, 72.1%, and 83.0% for MA, FGR, GDM, and PE, respectively. The findings suggest that promoter profiling of cfDNA may be used as a biological biomarker for predicting pregnancy complications at early gestational age.

Linking endotypes to omics profiles in difficult-to-control asthma using the diagnostic Chinese medicine syndrome differentiation algorithm

Objective: Patients with difficult-to-control asthma have difficulty breathing almost all of the time, even leading to life-threatening asthma attacks. However, only few diagnostic markers for this disease have been identified. We aimed to take advantage of unique Chinese medicine theories for phenotypic classification and to explore molecular signatures in difficult-to-control asthma. Methods: The Chinese medicine syndrome differentiation algorithm (CMSDA) is a syndrome-scoring classification method based on the Chinese medicine overall observation theory. Patients with difficult-to-control asthma were classified into Cold- and Hot-pattern groups according to the CMSDA. DNA methylation and metabolomic profiles were obtained using Infinium Human Methylation 450 BeadChip and gas chromatography-mass spectrometer. Subsequently, an integrated bioinformatics analysis was performed to compare those two patterns and identify Cold/Hot-associated candidates, followed by functional validation studies. Results: A total of 20 patients with difficult-to-control asthma were enrolled in the study. Ten were grouped as Cold and 10 as Hot according to the CMSDA. We identified distinct whole-genome DNA methylation and metabolomic profiles between Cold- and Hot-pattern groups. ALDH3A1 gene exhibited variations in the DNA methylation probe cg10791966, while two metabolic pathways were associated with those two patterns. Conclusions: Our study introduced a novel diagnostic classification approach, the CMSDA, for difficult-to-control asthma. This is an alternative way to categorize diverse syndromes and link endotypes with omics profiles of this disease. ALDH3A1 might be a potential biomarker for precision diagnosis of difficult-to-control asthma.

Development of a Sequence-Based Reference Physical Map of Pea (Pisum sativum L.).

Whole genome profiling (WGP) is a sequence-based physical mapping technology and uses sequence tags generated by next generation sequencing for construction of bacterial artificial chromosome (BAC) contigs of complex genomes. The physical map provides a framework for assembly of genome sequence and information for localization of genes that are difficult to find through positional cloning. To address the challenges of accurate assembly of the pea genome (∼4.2 GB of which approximately 85% is repetitive sequences), we have adopted the WGP technology for assembly of a pea BAC library. Multi-dimensional pooling of 295,680 BAC clones and sequencing the ends of restriction fragments of pooled DNA generated 1,814 million high quality reads, of which 825 million were deconvolutable to 1.11 million unique WGP sequence tags. These WGP tags were used to assemble 220,013 BACs into contigs. Assembly of the BAC clones using the modified Fingerprinted Contigs (FPC) program has resulted in 13,040 contigs, consisting of 213,719 BACs, and 6,294 singleton BACs. The average contig size is 0.33 Mbp and the N50 contig size is 0.62 Mbp. WGPTM technology has proved to provide a robust physical map of the pea genome, which would have been difficult to assemble using traditional restriction digestion based methods. This sequence-based physical map will be useful to assemble the genome sequence of pea. Additionally, the 1.1 million WGP tags will support efficient assignment of sequence scaffolds to the BAC clones, and thus an efficient sequencing of BAC pools with targeted genome regions of interest.

Abstract 21: Lipid Metabolites Are Associated With Epigenetic Aging Acceleration in the Coronary Artery Risk Development in Young Adults (CARDIA) Study

Background: Epigenetic aging measures including phenotypic aging (PA) and extrinsic epigenetic age acceleration (EEAA) have been associated with age-related diseases and conditions that include cardiovascular diseases, diabetes, and cancer. However, associations between epigenetic aging and lipids have not been widely investigated. Hypothesis: We hypothesized that adverse lipid metabolite profiles are associated with epigenetic age acceleration. Methods: We estimated measures of epigenetic age acceleration in 957 white and black participants from the CARDIA Study with whole-genome blood DNA methylation profiling by the Illumina EPIC array at Year 20 (Y20; ages 38-52). We used separate linear regression models to examine cross-sectional associations of PA and EEAA estimates with fasting triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) levels after adjusting for race, sex, education, alcohol intake, smoking status and physical activity. Results: Participants were 51% female (n=490) and 39% black (n=393) with mean chronological age 45±4 years. Compared with participants in the 1st tertile of TG, those in the 2nd tertile had 1.32 years older PA (95%CI 0.33-2.30) and 1.04 years older EEAA (95%CI 0.22-1.87); those in the 3rd tertile had 1.78 years older PA (95%CI 0.75-2.81) and 1.09 years older EEAA (95%CI 0.23-1.95). Compared to participants in the 1st tertile of HDL-C, those in the 3rd tertile had decreased EEAA by 1.21 years (95%CI -2.16--0.27). There were no significant differences of PA or EEAA among participants of LDL-C tertiles. (Figure 1.) Conclusions: Adverse lipid metabolites (TG and HDL-C) are associated cross-sectionally with accelerated epigenetic aging. Further studies are needed to understand the longitudinal associations between epigenetic aging and adverse lipid profiles as well as the potential mechanisms that underlie these associations.

Genome-wide miRNA profiling reinforces the importance of miR-9 in human papillomavirus associated oral and oropharyngeal head and neck cancer

Head and neck cancer is the sixth most common malignancy worldwide, predominantly developing from squamous cell epithelia (HNSCC). The main HNSCC risk factors are tobacco, excessive alcohol use, and the presence of human papillomavirus (HPV). HPV positive (+) cancers are etiologically different from other HNSCC and often show better prognosis. The current knowledge regarding HNSCC miRNA profiles is still incomplete especially in the context of HPV+ cancer. Thus, we analyzed 61 freshly collected primary oral (OSCC) and oropharyngeal (OPSCC) SCC samples. HPV DNA and RNA was found in 21% cases. The Illumina whole-genome small-RNA profiling by next-generation sequencing was done on 22 samples and revealed 7 specific miRNAs to HPV+ OSCC, 77 to HPV+ OPSCC, and additional 3 shared with both; 51 miRNAs were specific to HPV− OPSCC, 62 to HPV− OSCC, and 31 shared with both. The results for 9 miRNAs (miR-9, -21, -29a, -100, -106b, -143 and -145) were assessed by reverse transcription-quantitative polymerase chain reaction on the whole study population. The data was additionally confirmed by reanalyzing publicly available miRNA sequencing Cancer Genome Atlas consortium (TCGA) HNSCC data. Cell signaling pathway analysis revealed differences between HPV+ and HPV− HNSCC. Our findings compared with literature data revealed extensive heterogeneity of miRNA deregulation with only several miRNAs consistently affected, and miR-9 being the most likely HPV related miRNA.

Whole-genome methylation profiling of the retinal pigment epithelium of individuals with age-related macular degeneration reveals differential methylation of the SKI, GTF2H4, and TNXB genes

BackgroundAge-related macular degeneration (AMD) is a degenerative disorder of the central retina and the foremost cause of blindness. The retinal pigment epithelium (RPE) is a primary site of disease pathogenesis. The genetic basis of AMD is relatively well understood; however, this knowledge is yet to yield a treatment for the most prevalent non-neovascular disease forms. Therefore, tissue-specific epigenetic mechanisms of gene regulation are of considerable interest in AMD. We aimed to identify differentially methylated genes associated with AMD in the RPE and differentiate local DNA methylation aberrations from global DNA methylation changes, as local DNA methylation changes may be more amenable to therapeutic manipulation.MethodsEpigenome-wide association study and targeted gene expression profiling were carried out in RPE cells from eyes of human donors. We performed genome-wide DNA methylation profiling (Illumina 450k BeadChip array) on RPE cells from 44 human donor eyes (25 AMD and 19 normal controls). We validated the findings using bisulfite pyrosequencing in 55 RPE samples (30 AMD and 25 normal controls) including technical (n = 38) and independent replicate samples (n = 17). Long interspersed nucleotide element 1 (LINE-1) analysis was then applied to assess global DNA methylation changes in the RPE. RT-qPCR on independent donor RPE samples was performed to assess gene expression changes.ResultsGenome-wide DNA methylation profiling identified differential methylation of multiple loci including the SKI proto-oncogene (SKI) (p = 1.18 × 10−9), general transcription factor IIH subunit H4 (GTF2H4) (p = 7.03 × 10−7), and Tenascin X (TNXB) (p = 6.30 × 10−6) genes in AMD. Bisulfite pyrosequencing validated the differentially methylated locus cg18934822 in SKI, and cg22508626 within GTF2H4, and excluded global DNA methylation changes in the RPE in AMD. We further demonstrated the differential expression of SKI, GTF2H4, and TNXB in the RPE of independent AMD donors.ConclusionsWe report the largest genome-wide methylation analysis of RPE in AMD along with associated gene expression changes to date, for the first-time reaching genome-wide significance, and identified novel targets for functional and future therapeutic intervention studies. The novel differentially methylated genes SKI and GTF2H4 have not been previously associated with AMD, and regulate disease pathways implicated in AMD, including TGF beta signaling (SKI) and transcription-dependent DNA repair mechanisms (GTF2H4).

IRF8/miR-451a Regulates M-MDSC Differentiation <i>via</i> the AMPK/mTOR Signal Pathway During Lupus Development

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease. Myeloid-derived suppressor cells (MDSCs) have been found to be involved in the regulation of SLE development. However, little is known about the association between MDSC subsets and the factors that draw MDSCs into abnormal expansion. This study found that M-MDSCs increased in mice with pristane-induced lupus. Toll-like receptor (TLR)7 signal activation and high IFN-α level promoted M-MDSC accumulation in vitro. Moreover, both AMPK agonist metformin and two mTOR inhibitors INK128 and rapamycin inhibited the percentage of M-MDSCs in mice with pristane-induced lupus as well as in the TLR7- and IFN-α-induced bone marrow (BM) differentiation into MDSCs in vitro. In terms of mechanism, whole-genome transcriptome profiling was performed by RNA sequencing, revealing that the expression of the transcription factor IRF-8 was higher in M-MDSCs isolated from mice with pristane-induced lupus compared with control mice. IRF-8 was identified to be crucial for TLR7- and IFN-α-induced BM differentiation into MDSCs in vitro. Furthermore, IRF-8 was targeted by miR-451a in M-MDSC differentiation. Of note, metformin-modified M-MDSCs could relieve lupus symptoms in mice with pristane-induced lupus. The findings revealed a novel mechanism linking IRF8/miR-451a to M-MDSC differentiation via the AMPK/mTOR signal pathway during lupus development. This study might provide an important reference for SLE therapy by targeting M-MDSCs. Funding Statement: This work was supported by the National Natural Science Foundation of China (No. 31872732, 31570909), the Fundamental Research Funds of the Central Universities (No. 021414380342) and the Fund of State Key Laboratory of Trauma, Burns and Combined Injury (No. SKLKF201602). Declaration of Interests: The authors declare no commercial or financial conflicts of interest. Ethics Approval Statement: All procedures involving mice were approved by the institutional license for animal care and use based on the Animal Care Committee at Nanjing University.

Разработка программного комплекса WebMCOT для поиска совместно встречаемых ДНК-мотивов сайтов связывания транскрипционных факторов

Regulation of eukaryotic gene transcription is controlled by specific proteins transcription factors. Transcription factors bind certain regions of genomic DNA (binding sites or motives). Common action of two or more transcription factors is widespread mechanism of transcription factor action. Hence, the term ‘composite element’ implied two closely located and frequently occurred in genomic DNA motives. Composite elements are partitioned onto those with two overlapped motifs, or with these two motifs separated with a spacer. Currently, the chromatin immunoprecipitation high throughput approach ChIP-seq is used to locate binding sites for a certain “anchor” transcription factor in vivo in genomic scale. Thus, the search of composite elements with the help of ChIP-seq whole-genome transcription factor binding profiles is the actual bioinformatics issue. But existing approaches for prediction of composite elements on the basis of ChIP-seq data either omit an overlap of motifs (but require only a single ChIP-seq dataset) or consider an overlap of motifs (but require additional ChIP-seq data for a partner motif). But, ChIP-seq experiments are very expensive. In the Institute of Cytology and Genetics, MCOT program has been recently developed. It performs search of motifs taking into account their overlaps based on a single ChIP-seq dataset. MCOT is a console application and does not have many user friendly functions like data preparation and report generation. This work presents a web service WebMCOT for prediction of co-occurred DNA motifs in ChIP-seq data. WebMCOT consists of three parts: client, server, and worker. Software tools list, architecture and web interface are presented.

Pharmacogenetic and pharmacogenomic discovery strategies.

Genetic/genomic profiling at a single-patient level is expected to provide critical information for determining inter-individual drug toxicity and potential efficacy in cancer therapy. A better definition of cancer subtypes at a molecular level, may correspondingly complement such pharmacogenetic and pharmacogenomic approaches, for more effective personalized treatments. Current pharmacogenetic/pharmacogenomic strategies are largely based on the identification of known polymorphisms, thus limiting the discovery of novel or rarer genetic variants. Recent improvements in cost and throughput of next generation sequencing (NGS) are now making whole-genome profiling a plausible alternative for clinical procedures. Beyond classical pharmacogenetic/pharmacogenomic traits for drug metabolism, NGS screening programs of cancer genomes may lead to the identification of novel cancer-driving mutations. These may not only constitute novel therapeutic targets, but also effector determinants for metabolic pathways linked to drug metabolism. An additional advantage is that cancer NGS profiling is now leading to discovering targetable mutations, e.g., in glioblastomas and pancreatic cancers, which were originally discovered in other tumor types, thus allowing for effective repurposing of active drugs already on the market.

MEIS2 regulates endothelial to hematopoietic transition of human embryonic stem cells by targeting TAL1

BackgroundDespite considerable progress in the development of methods for hematopoietic differentiation, efficient generation of transplantable hematopoietic stem cells (HSCs) and other genuine functional blood cells from human embryonic stem cells (hESCs) is still unsuccessful. Therefore, a better understanding of the molecular mechanism underlying hematopoietic differentiation of hESCs is highly demanded.MethodsIn this study, by using whole-genome gene profiling, we identified Myeloid Ectopic Viral Integration Site 2 homolog (MEIS2) as a potential regulator of hESC early hematopoietic differentiation. We deleted MEIS2 gene in hESCs using the CRISPR/CAS9 technology and induced them to hematopoietic differentiation, megakaryocytic differentiation.ResultsIn this study, we found that MEIS2 deletion impairs early hematopoietic differentiation from hESCs. Furthermore, MEIS2 deletion suppresses hemogenic endothelial specification and endothelial to hematopoietic transition (EHT), leading to the impairment of hematopoietic differentiation. Mechanistically, TAL1 acts as a downstream gene mediating the function of MEIS2 during early hematopoiesis. Interestingly, unlike MEIS1, MEIS2 deletion exerts minimal effects on megakaryocytic differentiation and platelet generation from hESCs.ConclusionsOur findings advance the understanding of human hematopoietic development and may provide new insights for large-scale generation of functional blood cells for clinical applications.

Tight junction, mucin, and inflammasome-related molecules are differentially expressed in eosinophilic, mixed, and neutrophilic experimental asthma in mice.

Asthma is a chronic respiratory disease with marked clinical and pathophysiological heterogeneity. Specific pathways are thought to be involved in the pathomechanisms of different inflammatory phenotypes of asthma; however, direct invivo comparison has not been performed. We developed mouse models representing three different phenotypes of allergic airway inflammation-eosinophilic, mixed, and neutrophilic asthma via different methods of house dust mite sensitization and challenge. Transcriptomic analysis of the lungs, followed by the RT-PCR, western blot, and confocal microscopy, was performed. Primary human bronchial epithelial cells cultured in air-liquid interface were used to study the mechanisms revealed in the invivo models. By whole-genome transcriptome profiling of the lung, we found that airway tight junction (TJ), mucin, and inflammasome-related genes are differentially expressed in these distinct phenotypes. Further analysis of proteins from these families revealed that Zo-1 and Cldn18 were downregulated in all phenotypes, while increased Cldn4 expression was characteristic for neutrophilic airway inflammation. Mucins Clca1 (Gob5) and Muc5ac were upregulated in eosinophilic and even more in neutrophilic phenotype. Increased expression of inflammasome-related molecules such as Nlrp3, Nlrc4, Casp-1, and IL-1β was characteristic for neutrophilic asthma. In addition, we showed that inflammasome/Th17/neutrophilic axis cytokine-IL-1β-may transiently impair epithelial barrier function, while IL-1β and IL-17 increase mucin expressions in primary human bronchial epithelial cells. Our findings suggest that differential expression of TJ, mucin, and inflammasome-related molecules in distinct inflammatory phenotypes of asthma may be linked to pathophysiology and might reflect the differences observed in the clinic.

The Complexity of the Identification of Burkholderia cepacia Strain Which Caused Septicemia

Background: The correct identifying of pathogenic Burkholderia spp. using available commercial biochemical systems is a certain problem due to metabolic plasticity and variable enzymatic profile of isolates. Objectives: The current study aimed at using specific PCR and conventional multi-locus sequence typing (MLST) scheme to confirm the uncertain identification results for Burkholderia cepacia clinical isolate. Methods: Multilocus species-specific PCR and MLST profiling of high-throughput sequencing data have been used to clarify the varied results of biochemical identification of the strain. Results: The strain isolated from a patient with septicemia was initially identified as B. pseudomallei by Vitek 2 GN system but has an uncharacteristic antimicrobial resistance pattern and colony morphology. A species-specific multilocus PCR and whole-genome sequence profiling, according to the MLST scheme, allowed to identify an isolate as B. cepacia. Conclusions: The obtained results demonstrate the preference of molecular tests for correct identifying of pathogenic Burkholderia, considering that misidentification of those may lead to improper treatment or increase of biosafety risk.