Differential Expression Signatures Research Articles

Sexual reproduction during diatom bloom.

Phytoplankton supports food webs in all aquatic ecosystems. Ecological studies highlighted the links between environmental variables and species successions in situ. However, the role of life cycle characteristics on phytoplankton community dynamics remains poorly characterized. In diatoms, sexual reproduction creates new genetic combinations and prevents excessive cell size miniaturization. It has been extensively studied in vitro but seldom in the natural environment. Here, analyzing metatranscriptomic data in the light of the expression patterns previously characterized in vitro, we identified a synchronized and transient sexual reproduction event during a bloom of the toxic diatom species Pseudo-nitzschia australis. Despite the complexity of environmental conditions encountered in situ, sexual reproduction appeared to be the strongest differential gene expression signal that occurred during the bloom. The potential link between environmental conditions and the initiation of sexual reproduction remain to be determined, but sexual reproduction probably had a major impact on the bloom dynamic.

Single-nucleus transcriptomics reveals disease- and pathology-specific signatures in α-synucleinopathies.

α-synucleinopathies are progressive neurodegenerative disorders characterized by intracellular aggregation of α-synuclein, yet their molecular pathogenesis remains unknow. Here, we explore cell-specific changes in gene expression across different α-synucleinopathies. We perform single-nucleus RNA sequencing on nearly 300,000 nuclei from the prefrontal cortex of individuals with idiopathic Parkinson's disease (iPD, n = 20), Parkinson's disease caused by LRRK2 mutations (LRRK2-PD, n = 7), multiple system atrophy (MSA, n = 6) and healthy controls (n = 13). iPD and LRRK2-PD exhibit a largely overlapping cell type-specific signature, which is distinct from that of MSA, and includes an overall decrease of the transcriptional output in neurons. Notably, most of the differential expression signal in iPD and LRRK2-PD is concentrated in a specific deep cortical neuronal subtype expressing adrenoceptor alpha 2A. While most differentially expressed genes are highly cell type- and disease-specific, PDE10A is found consistently downregulated in most cortical neurons, and across all three diseases. Finally, exploiting the variable presence and/or severity of α-synuclein pathology in LRRK2-PD and iPD, we identify cell type-specific signatures associated with α-synuclein pathology, including a neuronal upregulation of the SNCA gene itself, encoding α-synuclein. Our findings provide novel insights into the cell-specific transcriptional landscape of the α-synucleinopathy spectrum.

Optimizing in silico drug discovery: simulation of connected differential expression signatures and applications to benchmarking.

We present a novel simulation method for generating connected differential expression signatures. Traditional methods have struggled with the lack of reliable benchmarking data and biases in drug-disease pair labeling, limiting the rigorous benchmarking of connectivity-based approaches. Our aim is to develop a simulation method based on a statistical framework that allows for adjustable levels of parametrization, especially the connectivity, to generate a pair of interconnected differential signatures. This could help to address the issue of benchmarking data availability for connectivity-based drug repurposing approaches. We first detailed the simulation process and how it reflected real biological variability and the interconnectedness of gene expression signatures. Then, we generated several datasets to enable the evaluation of different existing algorithms that compare differential expression signatures, providing insights into their performance and limitations. Our findings demonstrate the ability of our simulation to produce realistic data, as evidenced by correlation analyses and the log2 fold-change distribution of deregulated genes. Benchmarking reveals that methods like extreme cosine similarity and Pearson correlation outperform others in identifying connected signatures. Overall, our method provides a reliable tool for simulating differential expression signatures. The data simulated by our tool encompass a wide spectrum of possibilities to challenge and evaluate existing methods to estimate connectivity scores. This may represent a critical gap in connectivity-based drug repurposing research because reliable benchmarking data are essential for assessing and advancing in the development of new algorithms. The simulation tool is available as a R package (General Public License (GPL) license) at https://github.com/cgonzalez-gomez/cosimu.

Probabilistic association of differentially expressed genes with cis-regulatory elements.

Differential gene expression in response to perturbations is mediated at least in part by changes in binding of transcription factors (TFs) and other proteins at specific genomic regions. Association of these cis-regulatory elements (CREs) with their target genes is a challenging task that is essential to address many biological and mechanistic questions. Many current approaches rely on chromatin conformation capture techniques or single-cell correlational methods to establish CRE-to-gene associations. These methods can be effective but have limitations, including resolution, gaps in detectable association distances, and cost. As an alternative, we have developed DegCre, a nonparametric method that evaluates correlations between measurements of perturbation-induced differential gene expression and differential regulatory signal at CREs to score possible CRE-to-gene associations. It has several unique features, including the ability to use any type of CRE activity measurement, yield probabilistic scores for CRE-to-gene pairs, and assess CRE-to-gene pairings across a wide range of sequence distances. We apply DegCre to six data sets, each using different perturbations and containing a variety of regulatory signal measurements, including chromatin openness, histone modifications, and TF occupancy. To test their efficacy, we compare DegCre associations to Hi-C loop calls and CRISPR-validated CRE-to-gene associations, establishing good performance by DegCre that is comparable or superior to competing methods. DegCre is a novel approach to the association of CREs to genes from a perturbation-differential perspective, with strengths that are complementary to existing approaches and allow for new insights into gene regulation.

209 Transcriptomic Analysis of the Post-mortem Brain in Intracranial Atherosclerosis Implicates Interferon Signaling

INTRODUCTION: Intracranial atherosclerotic stenosis (ICAS) is a common cause of ischemic stroke. Stroke patients manifesting symptomatic ICAS often have poor prognoses, including worsened functional outcome, increased morbidity, and mortality. Despite risk factor management, ICAS exhibits a nonlinear rise in incidence with age, with an increase in unstable lipid-rich plaques. Understanding mechanisms of ICAS is crucial to improve clinical outcomes and reduce stroke incidence. Previous studies have demonstrated that intracranial arteries demonstrate different histopathologic profiles compared to extracranial arteries, likely lowering their risk of atherosclerosis. However, these studies are limited to healthy intracranial arteries. Comprehensive multiomic data examining diseased intracranial arteries are limited. METHODS: We performed bulk transcriptomic analysis in 4 tissues in a post-mortem brain dataset (N=304), with 34 patients with cerebral atherosclerosis confirmed via autopsy, 90 patients with peripheral atherosclerosis, and 180 controls. Cell type deconvolution was conducted to identify differential expression signatures in 7 neuronal and glial subtypes. To examine interferon response further in glial cells, we generated human induced pluripotent stem cell (hiPSC)-derived astrocytes treated with 3 doses of interferon and performed transcriptomic analysis. RESULTS: Cerebral atherosclerosis was significantly (pathway enrichment p<10e-9) associated with interferon signaling compared to peripheral atherosclerosis and controls. Glial cells particularly expressed patterns associated with cerebral atherosclerosis. Astrocytes, oligodendrocytes, excitatory neurons, and endothelial cells were most impacted. In hiPSC-derived astrocytes treated with interferon, we identified upregulation of interferon-inducible genes matching those found in intracranial atherosclerosis in the post-mortem brain, implicating glial reactivity to interferon-related stress. CONCLUSIONS: Our study implicates interferon signaling as critical within the development of cerebral atherosclerosis. Protection of the intracranial arteries from atherosclerosis may occur due to reduced expression of interferon-inducible genes. Targeted treatment of cerebral atherosclerosis should consider interferon-signaling modulators.

Abstract 3541: Systematic assessment of the mechanism of action landscape of glue compounds

Abstract We have recently completed a large-scale project to elucidate the Mechanism of Action (MoA) of clinically relevant compounds in over twenty tumor specific contexts. Many novel, translationally relevant findings have arisen from this work, including (a) the identification of context-specific inhibitors of many proteins considered to be poor pharmacologic targets—including transcription (co-)factors and undruggable signaling proteins; (b) graph theory-based elucidation of drug polypharmacology; and (c) identification of MoA overlap between previously unrelated drugs. However, a limitation of the study is it relies on the existing repertoire of small-molecular inhibitors that is heavily biased towards specific target classes. Compounds that inhibit master regulators (MR) of tumor state are missing or under-represented. In this new project, we explore an extra molecular space of “molecular glues”, as they might be more effective modulators of MRs. Similar to our prior oncology drug study, we will leverage the automated PLATE-Seq technology and the VIPER algorithm to characterize compound MoA in a proteome-wide fashion. Specifically, we will first generate genome-wide RNA-Seq profiles of patient-matched cell lines using a panel of ~150 glue compounds, including a subset of them named PROteolysis TArgeting Chimeras (PROTACs) that were designed to degrade therapeutic targets by recruiting them to E3 ligases. We will analyze the PLATE-seq perturbational profiles with a set of complementary algorithms including the VIPER, which, akin to a multiplexed, tissue-specific gene reporter assay, can accurately measure the differential activity of over 6,000 regulatory/signaling proteins, by assessing the enrichment of their transcriptional targets in differentially expressed genes. Current effort of this project is focused on six PDA cell lines including three MAPK-pathway dependent (M+) and three MAPK-pathway independent (M-) contexts, each one representing the gastrointestinal lineage state (GLS), morphogenic state (MOS), and acinar to ductal metaplasia-like state (ALS). Using this data, we explore similarity-based connections between PLATE-seq perturbational profiles and the Connectivity Map (CMAP) that contains transcriptional consequences of chemical and genetic perturbations. We will also compare PLATE-seq perturbational profiles to tissue-specific differential expression signatures that distinguish cell lines sensitive to compounds in the Cancer Therapeutics Response Portal (CTRP) and Broad Repurposing Hub, each of which houses results from exposing hundreds of compounds exposed to dozens to hundreds of cell lines. Taken together, the data and analyses will help understand MoAs and polypharmacologies/off-targets of molecular glues, which also serve to enable interpretation and prioritization of new candidate MoA for molecular glues by comparing them to compounds with known targets and MoAs. Citation Format: Lucas ZhongMing Hu, Charles Karan, Andrea Califano. Systematic assessment of the mechanism of action landscape of glue compounds [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3541.

Reappearance of CXCR4 dim/CD5 bright Proliferative Fraction Associates with BTK Mutations and Anticipates Progression in Ibrutinib-Treated CLL

Reciprocal expression of CXCR4 and CD5 on the surface of chronic lymphocytic leukemia (CLL) cells can discriminate the proliferative fraction (CD5high/CXCR4dim, PF), recently egressed from the lymph node, from the resting fraction (CXCR4high/CD5dim, RF) of older, quiescent cells. BTK inhibitors affect the proliferative capacity of CLL cells and cause the redistribution from nodal compartments to the blood stream. Nevertheless, little is known about the impact of long-term treatment of the BTK-inhibitor ibrutinib (IBR) on the PF/RF phenotype. Here, we monitored the PF dynamics in two cohorts of IBR-treated CLL cases with long-term follow-up, and correlated with the emergence of IBR resistance. Longitudinal analysis of the PF under IBR in 31 CLL cases, enrolled in the IOSI-EMA-001 with sequential blood samplings (156 samples, median 6 samples/case) showed a depletion of the PF over time, from 17.7% at pre-treatment to 4.75, 0.4, 1.5, 0.6, 1.5% at later timepoints (0.5, 6, 12, 18, 24 months; p < 0.001, U test), along with loss of proliferative potential by Ki67 expression. We then retrospectively analyzed the PF in 100 IBR-treated CLL cases from the real world, referred for routine immunophenotyping (296 samples, median 3 samples/case). Median PF of pre-IBR samples was 12.0% (range 0.7-50), with a significant drop to 2.7% within one year of treatment and to 1.0% in the second year (p<0.001, U test). 77 cases discontinued IBR due to: toxicity (n=13), progression (n=51), other/death (n=13). Mutations of BTK/ PLCG2 were detected in 30 cases, 28 of which discontinued IBR due to progression and 2 for other/death. To correlate reappearance of the PF with cause of discontinuation, we focused on 64 cases collected within 12 months before/after IBR discontinuation due to toxicity (n=10), progression (n=47) or other/death (n=7), and compared with 22 samples still on treatment for the next 12 months after testing (Panel A). ROC analysis of the PF suggested a criterion of PF>3% as the most discriminating to predict IBR discontinuation due to progression, with a sensitivity of 86.7% and a specificity of 71.1%. This cut-off could separate patients with a longer time-to-progression (median 56 vs. 45 months, p=0.031). 26/47 progressing cases bore BTK/ PLCG2 mutations, with a PF higher than wild-type cases discontinuing for toxicity (8.4% vs. 2.2%; p<0.001) or still on treatment (1.8%, p<0.001). Notably, the 21 BTK/ PLCG2- wild-type progressing cases also showed higher PF compared to cases discontinuing for toxicity (6.9% vs. 2.2%; p<0.001) or still on treatment (1.8%; p<0.001). Notably, PF levels showed no significant correlation with white blood cell counts. Sequential dosages of plasmatic B2M in progressing cases (n=5) revealed steady levels below the 3.5 ug/mL threshold, despite the PF increase (median PF 18%). Cell sorting of the PF/RF fractions from 10 cases showing PF reappearance after prolonged IBR treatment (median 50.7 months) revealed a mean of 1.7 BTK mutations per sample (27 total, range 1-4), a median VAF higher in the PF (23.0%, 0.3-92.3) on average 2.8 times larger than in the RF (5.2%, 0.1-84.8; p=0.003, paired rank test). A similar skewing was present for PLCG2 mutations (median PF VAF: 2.0%, 0.1-6.2; RF VAF: 1.35%, 0.4-2.8; p=0.014). Finally, mRNA-seq was performed in 20 cases, on matching PF/RF fractions both at pre-IBR and progression (9 cases with BTK mutations). Differential expression signature of pre-IBR PF vs. RF, concordant with published gene sets, drove co-clustering of post-IBR PF/RF with their respective counterparts, indicating that PF at progression functionally resembles its pre-treatment counterpart. A strong enrichment of multiple proliferation-related signatures, present in pre-treatment samples, was significantly up-regulated also at progression, independently from BTK mutations (Panel B). In summary, here we report that CLL progression under IBR associates with reappearance of the CD5bright/CXCR4dim PF which precedes clinical progression, increase of serum B2M and WBC counts. Transcriptomic/genetic profiling of the post-IBR PF suggests presence of clonal selection dynamics and active bypass signaling pathways, bona fide taking place within the lymph node microenvironment, including BTK/ PLCG2 mutations. Clinically, longitudinal monitoring of the CXCR4/CD5 fractions by flow cytometry may provide a simple tool helping to intercept CLL progression under IBR therapy.

Deciphering Potential Molecular Signatures to Differentiate Acute Myeloid Leukemia (AML) with BCR::ABL1 from Chronic Myeloid Leukemia (CML) in Blast Crisis.

Acute myeloid leukemia (AML) with BCR::ABL1 has recently been recognized as a distinct subtype in international classifications. Distinguishing it from myeloid blast crisis chronic myeloid leukemia (BC-CML) without evidence of a chronic phase (CP), remains challenging. We aimed to better characterize this entity by integrating clonal architecture analysis, mutational landscape assessment, and gene expression profiling. We analyzed a large retrospective cohort study including CML and AML patients. Two AML patients harboring a BCR::ABL1 fusion were included in the study. We identified BCR::ABL1 fusion as a primary event in one patient and a secondary one in the other. AML-specific variants were identified in both. Real-time RT-PCR experiments demonstrated that CD25 mRNA is overexpressed in advanced-phase CML compared to AML. Unsupervised principal component analysis showed that AML harboring a BCR::ABL1 fusion was clustered within AML. An AML vs. myeloid BC-CML differential expression signature was highlighted, and while ID4 (inhibitor of DNA binding 4) mRNA appears undetectable in most myeloid BC-CML samples, low levels are detected in AML samples. Therefore, CD25 and ID4 mRNA expression might differentiate AML with BCR::ABL1 from BC-CML and assign it to the AML group. A method for identifying this new WHO entity is then proposed. Finally, the hypothesis of AML with BCR::ABL1 arising from driver mutations on a BCR::ABL1 background behaving as a clonal hematopoiesis mutation is discussed. Validation of our data in larger cohorts and basic research are needed to better understand the molecular and cellular aspects of AML with a BCR::ABL1 entity.

Cross-Comparison of Inflammatory Skin Disease Transcriptomics Identifies PTEN as a Pathogenic Disease Classifier in Cutaneous Lupus Erythematosus

Tissue transcriptomics is used to uncover molecular dysregulations underlying diseases. However, the majority of transcriptomics studies focus on single diseases with limited relevance for understanding the molecular relationship between diseases or for identifying disease-specific markers. In this study, we used a normalization approach to compare gene expression across nine inflammatory skin diseases. The normalized datasets were found to retain differential expression signals that allowed unsupervised disease clustering and identification of disease-specific gene signatures. Using the NS-Forest algorithm, we identified a minimal set of biomarkers and validated their use as diagnostic disease classifier. Among them, PTEN was identified as being a specific marker for cutaneous lupus erythematosus and found to be strongly expressed by lesional keratinocytes in association with pathogenic type I IFNs. In fact, PTEN facilitated the expression of IFN-β and IFN-κ in keratinocytes by promoting activation and nuclear translocation of IRF3. Thus, cross-comparison of tissue transcriptomics is a valid strategy to establish a molecular disease classification and to identify pathogenic disease biomarkers.

A panel of TDP-43-regulated splicing events verifies loss of TDP-43 function in amyotrophic lateral sclerosis brain tissue

TDP-43 dysfunction is a molecular hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A major hypothesis of TDP-43 dysfunction in disease is the loss of normal nuclear function, resulting in impaired RNA regulation and the emergence of cryptic exons. Cryptic exons and differential exon usage are emerging as promising markers of lost TDP-43 function in addition to revealing biological pathways involved in neurodegeneration in ALS/FTD. In this brief report, we identified markers of TDP-43 loss of function by depleting TARDBP from post-mortem human brain pericytes, a manipulable in vitro primary human brain cell model, and identifying differential exon usage events with bulk RNA-sequencing analysis. We present these data in an interactive database (https://www.scotterlab.auckland.ac.nz/research-themes/tdp43-lof-db-v2/) together with seven other TDP-43-depletion datasets we meta-analysed previously, for user analysis of differential expression and splicing signatures. Differential exon usage events that were validated by qPCR were then compiled into a ‘differential exon usage panel’ with other well-established TDP-43 loss-of-function exon markers. This differential exon usage panel was investigated in ALS and control motor cortex tissue to verify whether, and to what extent, TDP-43 loss of function occurs in ALS. We find that profiles of TDP-43-regulated cryptic exons, changed exon usage and changed 3′ UTR usage discriminate ALS brain tissue from controls, verifying that TDP-43 loss of function occurs in ALS. We propose that TDP-43-regulated splicing events that occur in brain tissue will have promise as predictors of disease.

Downregulation of monocyte miRNAs: implications for immune dysfunction and disease severity in drug-resistant tuberculosis.

Monocyte miRNAs govern both protective and pathological responses during tuberculosis (TB) through their differential expression and emerged as potent targets for biomarker discovery and host-directed therapeutics. Thus, this study examined the miRNA profile of sorted monocytes across the TB disease spectrum [drug-resistant TB (DR-TB), drug-sensitive TB (DS-TB), and latent TB] and in healthy individuals (HC) to understand the underlying pathophysiology and their regulatory mechanism. We sorted total monocytes including three subsets (HLA-DR+CD14+, HLA-DR+CD14+CD16+, and HLA-DR+CD16+cells) from peripheral blood mononuclear cells (PBMCs) of healthy and TB-infected individuals through flow cytometry and subjected them to NanoString-based miRNA profiling. The outcome was the differential expression of 107 miRNAs particularly the downregulation of miRNAs in the active TB groups (both drug-resistant and drug-sensitive). The miRNA profile revealed differential expression signatures: i) decline of miR-548m in DR-TB alone, ii) decline of miR-486-3p in active TB but significant elevation only in LTB iii) elevation of miR-132-3p only in active TB (DR-TB and DS-TB) and iv) elevation of miR-150-5p in DR-TB alone. The directionality of functions mediated by monocyte miRNAs from Gene Set Enrichment Analysis (GSEA) facilitated two phenomenal findings: i) a bidirectional response between active disease (activation profile in DR-TB and DS-TB compared to LTB and HC) and latent infection (suppression profile in LTB vs HC) and ii) hyper immune activation in the DR-TB group compared to DS-TB. Thus, monocyte miRNA signatures provide pathological clues for altered monocyte function, drug resistance, and disease severity. Further studies on monocyte miRNAs may shed light on the immune regulatory mechanism for tuberculosis.

Axonal Guidance Dysregulation in Hippocampus underlines a three-hit model of stress susceptibility

Under some combinations of genetic components (hit-1) and early-life conditions (hit-2), the developing phenotype gets compromised when exposed to challenges later in life (hit-3), enhancing the risk for psychiatric symptoms. We tested this “three-hit” hypothesis in a rat line that was genetically selected for high apomorphine-susceptibility (APO-SUS) as the first-hit using Wistar rats as controls. Pups that received low maternal care, as judged from low maternal licking and grooming (LG) scores, were considered to suffer from the second-hit, compared to pups receiving high maternal-LG. As a third-hit, some rats were exposed to post-weaning isolation-rearing, with social-rearing as the control condition. All adult APO-SUS rats displayed high levels of apomorphine-induced gnawing (p<.001; n=120/strain). The APO-SUS Low-LG offspring displayed enhanced hormonal response to stress (p<.001 for ACTH and corticosterone; n=15/maternal care group) and low acoustic startle (p<.050; n=30/maternal care group). The APO-SUS Low-LG offspring that were reared in isolation displayed low acoustic startle and a severe deficit in pre-pulse-inhibition (PPI), which was restored rapidly by CORT-treatment (p<.001 for both; n=10/LG by rearing group). The differential-expression signature in hippocampus (95 genes FDR5%) was enriched with neuron-specific genes (p<.001) and suggested altered axonal guidance (B-H p<.001). Human hippocampal expression signatures of childhood trauma survivors with and without genetic susceptibility for stress are concordant with the rat study. We conclude that cumulative stressful experience during development in genetically-predisposed individuals precipitates behavioral alterations associated with altered stress responsiveness and compromised hippocampus function.

MARSY: a multitask deep-learning framework for prediction of drug combination synergy scores.

Combination therapies have emerged as a treatment strategy for cancers to reduce the probability of drug resistance and to improve outcomes. Large databases curating the results of many drug screening studies on preclinical cancer cell lines have been developed, capturing the synergistic and antagonistic effects of combination of drugs in different cell lines. However, due to the high cost of drug screening experiments and the sheer size of possible drug combinations, these databases are quite sparse. This necessitates the development of transductive computational models to accurately impute these missing values. Here, we developed MARSY, a deep-learning multitask model that incorporates information on the gene expression profile of cancer cell lines, as well as the differential expression signature induced by each drug to predict drug-pair synergy scores. By utilizing two encoders to capture the interplay between the drug pairs, as well as the drug pairs and cell lines, and by adding auxiliary tasks in the predictor, MARSY learns latent embeddings that improve the prediction performance compared to state-of-the-art and traditional machine-learning models. Using MARSY, we then predicted the synergy scores of 133722 new drug-pair cell line combinations, which we have made available to the community as part of this study. Moreover, we validated various insights obtained from these novel predictions using independent studies, confirming the ability of MARSY in making accurate novel predictions. An implementation of the algorithms in Python and cleaned input datasets are provided in https://github.com/Emad-COMBINE-lab/MARSY.

Expression characteristics of circular RNA in human traumatic brain injury.

Traumatic brain injury (TBI) causes high rates of worldwide mortality and morbidity due to the complex secondary injury cascade. Recently, circular ribonucleic acids (circRNAs) have attracted significant attention in a variety of diseases. However, their expression characteristics in human TBI are still unclear. In this study, we examined brain injury tissues from six severe TBI patients in Jinling Hospital. The TBI tissues and adjacent brain contusion tissues were used to analyze differential expression signatures of circRNAs through full-length transcriptome sequencing, Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and ceRNA network construction. Our results found that there were 126 differently expressed circRNAs in TBI. Among them, 64 circRNAs were up-regulated and 62 circRNAs were down-regulated. Moreover, GO and KEGG analyses revealed that the aberrantly expressed circRNAs participated in many pathophysiological processes of TBI, especially regarding microglial cell activation, protein transport, protein processing and inflammation. Furthermore, the ceRNA (circRNA-miRNA-mRNA) network predicted that there existed strong relationship among circRNA, miRNA and mRNA. Taken together, our results indicated for the first time that the expression profiles of circRNAs were different after human TBI. In addition, we found the signaling pathways that were related to circRNAs and predicted a ceRNA network, which provided new insight of circRNAs in human TBI.

The CXCR4dim/CD5bright Proliferative Fraction Reappears in Relapsed CLL Under Ibrutinib, Enriched in BTK Mutations

BTK inhibitors deeply affect the proliferative capacity, homing and migrations of chronic lymphocytic leukemia (CLL) cells, and cause their redistribution from nodal compartments to the blood stream. Reciprocal expression of CXCR4 and CD5 on circulating CLL cells is an established marker discriminating the proliferative fraction (CD5high/CXCR4dim, PF), recently egressed from the lymph node, from the resting fraction (CXCR4high/CD5dim, RF) of older, quiescent cells navigating the blood stream. We monitored the dynamics of the PF under the course of ibrutinib therapy in 31 CLL cases recruited in the IOSI-EMA-001 study (NCT02827617) with sequential blood sampling (156 samples, median 6 samples/case, range 3-7). PF was monitored through fixed gates, designed on the pre-treatment sample around the population visually detaching from the main cluster (panel A). Longitudinal analysis showed a depletion of the PF over time, from a median of 17.7% at pre-treatment to 4.75, 0.4, 1.5, 0.6, 1.5% at subsequent timepoints (0.5, 6, 12, 18, 24 months; range 0-50; p < 0.001, U test). Loss of proliferative potential was confirmed by concurrent loss of Ki67 expression. We retrospectively analyzed the PF from a second cohort of 99 ibrutinib-treated CLL cases from the real world, referred for routine immunophenotyping (278 samples, median 3 samples/case, range 2-7). Median PF of pre-ibrutinib samples was 12.0% (range 0.7-50%), with a significant drop to 2.7% after 1 year of treatment and to 1.0% after 2 years (p<0.001, U test). Out of 99 cases, 55 discontinued ibrutinib within 5 years (16 toxicity, 38 progression/relapse, 1 death; median time of treatment 29.7 months, range 1-58.6 months). Median PF in samples collected after ibrutinib discontinuation was 1.15% after 2 years of treatment, and increased to 13.3, 7.10, 6.30, 12.0% after 3, 4, 5 and >5 years. Conversely, samples collected under ibrutinib treatment presented a more stable trend of median PF (3.0 and 3.5% after 3 and 4 years respectively). Interestingly, of 27 patients with blood samples collected during the third year of therapy, 11 failed ibrutinib due to relapse/progression within the following two years: these cases presented a higher median PF (10.0%) than cases who stayed on ibrutinib (median PF 1.8%, p=0.045). Mutations of BTK/PLCG2 were detected in 28/99 cases (28.2%); among these, 18 (64.2%) discontinued ibrutinib due to progression/relapse (17/18) or toxicity (1/18), whereas 10 cases, although mutated, remained on treatment. Median time of appearance of BTK/PLCG2 mutations was 48 months (range 16-70). Median VAF was 22.8 (range 1.2-99.0) and 3.5 (range 1.7-17.6) for BTK and PLCG2, respectively. We isolated the PF and RF fractions from 10 cases developing BTK/PLCG2 mutations, who showed reappearance of the PF after prolonged ibrutinib treatment (median 50.7 months). Sequencing revealed 1.7 mean BTK mutations per sample (range 1-4), with a median VAF higher in the PF (23.0%, range 0.3-92.3) than in the RF (5.15%, range 0.1-84.8; p=0.0027, paired rank test); overall, the PF VAF was almost 3 times larger than the RF (average 2.82, range 0.01-38). Accompanying PLCG2 mutations were present in 6 cases, generally more frequent (average 3.7 mutations per case, range 1-8) but with lower burden (median VAF 1.7%, range 0.1-4.3) than BTK mutations; again, average the PF fraction had higher VAF than the RF (average PF: 2.0, range 0.1-6.2; average RF: 1.35, range 0.4-2.8; p=0.0142) (panel B). Finally, we performed mRNA-seq on 6 cases relapsed after ibrutinib, isolating the RF and the newly reconstituted PF, the latter enriched in BTK mutations, with matching fractions isolated from pre-ibrutinib samples. Differential expression signature of PF vs. RF at pre-treatment was concordant with published gene sets (e.g. Calissano et al. Mol Med 2011); application of this signature to post-ibrutinib PF/RF fractions drove co-clustering of 12 out of 13 fractions with their respective pre-treatment counterparts, suggesting that the reconstituted PF fraction at relapse is functionally similar to the original pre-treatment PF. Overall, our data support the hypothesis that, in patients undergoing ibrutinib treatment, BTK/PLCG2 mutations arise within the lymph nodes, the proliferative compartment of CLL; clinically, longitudinal monitoring of the CXCR4/CD5 fractions by flow cytometry may provide a simple tool helping to intercept CLL progression under ibrutinib therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Integration of ATAC-seq and RNA-seq analysis identifies key genes affecting intramuscular fat content in pigs.

Meat quality is one of the most important economic traits in pig breeding and production, and intramuscular fat (IMF) content is the major factor in improving meat quality. The IMF deposition in pigs is influenced by transcriptional regulation, which is dependent on chromatin accessibility. However, how chromatin accessibility plays a regulatory role in IMF deposition in pigs has not been reported. Xidu black is a composite pig breed with excellent meat quality, which is an ideal research object of this study. In this study, we used the assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing (RNA-seq) analysis to identify the accessible chromatin regions and key genes affecting IMF content in Xidu black pig breed with extremely high and low IMF content. First, we identified 21,960 differential accessible chromatin peaks and 297 differentially expressed genes. The motif analysis of differential peaks revealed several potential cis-regulatory elements containing binding sites for transcription factors with potential roles in fat deposition, including Mef2c, CEBP, Fra1, and AP-1. Then, by integrating the ATAC-seq and RNA-seq analysis results, we found 47 genes in the extremely high IMF (IMF_H) group compared with the extremely low IMF (IMF_L) group. For these genes, we observed a significant positive correlation between the differential gene expression and differential ATAC-seq signal (r2 = 0.42). This suggests a causative relationship between chromatin remodeling and the resulting gene expression. We identified several candidate genes (PVALB, THRSP, HOXA9, EEPD1, HOXA10, and PDE4B) that might be associated with fat deposition. Through the PPI analysis, we found that PVALB gene was the top hub gene. In addition, some pathways that might regulate fat cell differentiation and lipid metabolism, such as the PI3K-Akt signaling pathway, MAPK signaling pathway, and calcium signaling pathway, were significantly enriched in the ATAC-seq and RNA-seq analysis. To the best of our knowledge, our study is the first to use ATAC-seq and RNA-seq to examine the mechanism of IMF deposition from a new perspective. Our results provide valuable information for understanding the regulation mechanism of IMF deposition and an important foundation for improving the quality of pork.

High resolution single cell analysis of the developing human heart

Abstract The human heart is composed of various cell types with distinct embryonic origins and functions. Having an understanding of the components of the developing heart at a molecular level should provide better insights into its function in health and disease. Current single cell studies of human fetal hearts are limited by low cell numbers and poor cell type resolution. Here, we aimed to increase the number of cells studied while adding a spatial resolution. We analysed human hearts aged between post conception weeks of 8 and 11, where apex and base in each were processed, providing spatial resolution on cells constituting the myocardium and the great vessels. Our analysis suggests 6 main cell types in the developing hearts with a total of 28 subtypes and provides a differential expression signature for similar cell types with different origins and anatomical locations, including the smooth muscle cells and endothelial cells of the myocardium and the great vessels. Through immunohistochemistry, we validated our findings on these subtypes, as well as on the cells that were less clearly described before, such sinoatrial node cells or resident macrophages. Cell to cell interaction analysis inferred distinct molecular crosstalk across subtypes, including the cells of the great vessels or the vessels of the myocardium. Our findings suggest a list of transcription factors that might be involved in governing the identity of these cells. Collectively, our analysis provides a better understanding of human heart development, and will inform studies into disease modelling and disease progression. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation

Expression Analysis of Moritella viscosa-Challenged Atlantic Salmon Identifies Disease-Responding Genes, MicroRNAs and Their Predicted Target Genes and Pathways.

Moritella viscosa is a bacterial pathogen causing winter-ulcer disease in Atlantic salmon. The lesions on affected fish lead to increased mortality, decreased fish welfare, and inferior meat quality in farmed salmon. MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional regulation by guiding the miRNA-induced silencing complex to specific mRNA transcripts (target genes). The goal of this study was to identify miRNAs responding to Moritella viscosa in salmon by investigating miRNA expression in the head-kidney and the muscle/skin from lesion sites caused by the pathogen. Protein coding gene expression was investigated by microarray analysis in the same materials. Seventeen differentially expressed guide-miRNAs (gDE-miRNAs) were identified in the head-kidney, and thirty-nine in lesion sites, while the microarray analysis reproduced the differential expression signature of several thousand genes known as infection-responsive. In silico target prediction and enrichment analysis suggested that the gDE-miRNAs were predicted to target genes involved in immune responses, hemostasis, angiogenesis, stress responses, metabolism, cell growth, and apoptosis. The majority of the conserved gDE-miRNAs (e.g., miR-125, miR-132, miR-146, miR-152, miR-155, miR-223 and miR-2188) are known as infection-responsive in other vertebrates. Collectively, the findings indicate that gDE-miRNAs are important post-transcriptional gene regulators of the host response to bacterial infection.

Circulating microRNA expression profiles to identify a potential link between prostate cancer and obesity

Effective diagnostic methods are needed to apply appropriate treatment strategies in patients with aggressive prostate cancer. From this point of view, risk factors that cause prostate cancer or its aggressiveness should be considered. Obesity is a critical risk factor for triggering prostate cancer's metastatic properties. microRNAs are used as biomarkers in diagnosing cancer and obesity depending on their tissue-specific expression patterns. This study investigates the role of obesity in the metastatic profile of prostate cancer depending on the differential expression signatures of selected miRNAs in prostate cancer and obese patients. The roles of miR-100, miR-141 and miR-145 in prostate cancer and obesity are partially known. However, their potential to become circular biomarkers in the blood is not elucidated. There is no previous data on miR-4463 and miR-653 on prostate cancer and obesity association. In this study, the blood samples were taken and obtained serum from 69 patients of 6 subgroups that consisted of one healthy group and five unhealthy groups based on their different prostate cancer or obesity levels. Five selected miRNA expression analyses (miR-100, miR-141, miR-145, miR-4463, and miR-653) were performed through total RNA isolation, which was confirmed via synthetic cel-miR-39 miRNA. Quantitative Real-Time PCR analyzed the expression levels of selected miRNAs. Data analysis was performed via normalising target miRNA expression levels with cel-miR-39. In this study, we found that the relationship between prostate cancer and obesity was investigated at the molecular level. It was suggested that target miR-100 could be a promising biomarker for non-obese and aggressive prostate cancer patients. miR-145 is a more potential biomarker than miR-141 for non-aggressive and non-obese patients. miR-4463 can be used to predict more prostate cancer patients than obese patients. Lastly, miR-653 can be a biomarker for non-aggressive prostate cancer cells.

Abstract P2064: High Resolution Single Cell Analysis Of The Developing Human Heart

The human heart is composed of various cell types with distinct embryonic origins and functions. Having an understanding of the components of the developing heart at a molecular level should provide better insights into its function in health and disease. Current single cell studies of human fetal hearts are limited by low cell numbers and poor cell type resolution. Here, we aimed to increase the number of cells studied while adding a spatial resolution. We analysed human hearts aged between post conception weeks of 8 and 11, where apex and base in each were processed, providing spatial resolution on cells constituting the myocardium and the great vessels. Our analysis suggests 6 main cell types in the developing hearts with a total of 28 subtypes and provides a differential expression signature for similar cell types with different origins and anatomical locations, including the smooth muscle cells and endothelial cells of the myocardium and the great vessels. Through immunohistochemistry, we validated our findings on these subtypes, as well as on the cells that were less clearly described before, such sinoatrial node cells or resident macrophages. Cell to cell interaction analysis inferred distinct molecular crosstalk across subtypes, including the cells of the great vessels or the vessels of the myocardium. Our findings suggest a list of transcription factors that might be involved in governing the identity of these cells. Collectively, our analysis provides a better understanding of human heart development, and will inform studies into disease modelling and disease progression.