Gene Expression Fingerprint Research Articles

Stromal cells engineered to express T cell factors induce robust CLL cell proliferation in vitro and in PDX co-transplantations allowing the identification of RAF inhibitors as anti-proliferative drugs

Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.

Abstract 2781: GBM cells mimic regulatory T cell function to protect the CSC pool from immune surveillance in recurrent GBM

Abstract Glioblastoma (GBM) is the most lethal and aggressive form of brain cancer, with incredibly high recurrence rates and virtually no long-term survival. Despite our growing understanding of GBM at the molecular level, we still don’t fully comprehend the molecular mechanisms driving recurrence in GBM. Current knowledge indicates that Glioma Stem Cells (GSCs) drive a resistant cell phenotype in GBM. Understanding the molecular events coordinated by GSCs leading to therapy-resistance can provide key advancements on how we treat recurrent GBM (rGBM). Mechanisms of GSC immune escape are considered fundamental to clinical GBM growth and recurrence. The cross talk between cancer cells and the immune system is now classified as a hallmark of cancer. The current dogma is that cancer cells, including GSCs, influence recruitment of immune-suppressive cell subsets to tumor tissue, however how subsets of GSCs mimic this immune-suppressive effect within the tumor microenvironment remains to be elucidated. Single-cell RNA sequencing analysis of GBM neurospheres revealed a previously unrecognized Oct4/Sox2high/FOXP3− cell subpopulation with high expression of TGFβ1, CD39, CD73, PD-L1, and Galectin-1, a gene expression fingerprint typically associated with regulatory T cell (Tregs) and their immune suppressive functions within the tumor microenvironment. Bioinformatics analysis of public databases shows that the above-mentioned genes are enriched in the mesenchymal GBM subtype and highly correlated with TGFβ type II receptor (TGFBR2) expression in clinical GBM. Mechanistically, we show that blocking TGFBR2 or XBP1 signaling, key intermediary of this process, counteracts the immune-suppressive phenotype of rGBM cells by restoring CD4 and CD8 tumor killing capacity and reversing exhaustion in co-culture experiments. siRNA-mediated knock-down of TGFBR2 in rGBM cells also decreased CD206 expression and reduced release of anti-inflammatory chemokines in THP1 cells. By combining miRNA-based network analysis and advanced nanoparticle formulation for miRNA delivery we show that miR-16/124-3p effectively target all nodes of this immunosuppressive axis successfully blocking the immunosuppressive nature of rGBM cells. This research provides the first description of such neoplastic cells in any malignancy and has high potential translational impact since targeting these tumor cell subsets and their immunosuppressive mechanisms may be critical to the successful development of GBM immunotherapies. Citation Format: Hernando Lopez-Bertoni, Sophie Sall, Harmon Khela, Jack Korleski, Katherine Luly, Maya Johnson, Bachchu Lal, Amanda Johnson, Stephany Tzeng, Jordan Green, John Laterra. GBM cells mimic regulatory T cell function to protect the CSC pool from immune surveillance in recurrent GBM [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 2781.

Abstract B009: GBM cells mimic regulatory T cell function to protect the CSC pool from immune surveillance in recurrent GBM

Abstract Glioblastoma (GBM) is the most lethal and aggressive form of brain cancer, with incredibly high recurrence rates and virtually no long-term survival. Despite our growing understanding of GBM at the molecular level, we still don’t fully comprehend the molecular mechanisms driving recurrence in GBM. Current knowledge indicates that Glioma Stem Cells (GSCs) drive a resistant cell phenotype in GBM. Understanding the molecular events coordinated by GSCs leading to therapy-resistance can provide key advancements on how we treat recurrent GBM (rGBM). Mechanisms of GSC immune escape are considered fundamental to clinical GBM growth and recurrence. The cross talk between cancer cells and the immune system is now classified as a hallmark of cancer. The current dogma is that cancer cells, including GSCs, influence recruitment of immune-suppressive cell subsets to tumor tissue, however how subsets of GSCs mimic this immune-suppressive effect within the tumor microenvironment remains to be elucidated. Single-cell RNA sequencing analysis of GBM neurospheres revealed a previously unrecognized Oct4/Sox2high/FOXP3− cell subpopulation with high expression of TGFb1, CD39, CD73, PD-L1, and Galectin-1, a gene expression fingerprint typically associated with regulatory T cell (Tregs) and their immune suppressive functions within the tumor microenvironment. Bioinformatics analysis of public databases shows that the above-mentioned genes are enriched in the mesenchymal GBM subtype and highly correlated with TGFb type II receptor (TGFBR2) expression in clinical GBM. Mechanistically, we show that blocking TGFBR2 or XBP1 signaling, key intermediary of this process, counteracts the immune-suppressive phenotype of rGBM cells by restoring CD4 and CD8 tumor killing capacity and reversing exhaustion. By combining miRNA-based network analysis and advanced nanoparticle formulation for miRNA delivery we show that miR-16/124-3p effectively target all nodes of this immunosuppressive axis successfully blocking the immunosuppressive nature of rGBM cells. This research provides the first description of such neoplastic cells in any malignancy and has high potential translational impact since targeting these tumor cell subsets and their immunosuppressive mechanisms may be critical to the successful development of GBM immunotherapies. Citation Format: Hernando Lopez-Bertoni, Sophie Sall, Harmon Khela, Jack Korleski, Katherine Luly, Maya Johnson, Amanda Johnson, Jordan Green, John Laterra. GBM cells mimic regulatory T cell function to protect the CSC pool from immune surveillance in recurrent GBM [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B009.

Of mice and men: transcriptome-based clustering reveals molecular heterogeneity among mouse atherosclerosis models and partially reflects the diversity of human plaques

Abstract Background Mice are frequently used as model organisms to study atherosclerosis, but many treatments effective in mice do not successfully translate to humans. In a recent study, we clustered 654 human plaques based on transcriptomic data and described the categorisation into five distinct plaque types. We now hypothesise that the reasons for the failures of translating results from mouse models can be partially explained by the fact that mouse models do not reflect all the phenotypic variability we observe in human plaques. Methods We used transcriptomic data from the aortic samples (from arch to mid abdomen) of 102 different strains of inbred mice (n = 366). Those mice are F1 progenies from the Hybrid Mouse Diversity Panel (HMDP) crossed with C57BL/6J carrying dominant ApoE-Leiden and CETP transgenes. The gene expression data were normalised and batch effect was removed and clustered by a shared nearest neighbour modularity optimization-based clustering algorithm. Differential gene expression between the clusters was performed to reveal the difference in the biology of the clusters. The clusters were linked to physiological measurements and correlated to human transcriptomic-based plaque clusters. Results Transcriptional profiles of atherosclerotic lesions in mice could be categorised into four distinct clusters with specific gene expression fingerprints. The different clusters have a different activity for some pathways, including smooth muscle cell related pathways and metabolic pathways. Comparison with human clusters revealed that most mice strains (n = 287, 78%) correlated best with human plaque type #3 which is enriched in both inflammatory and fibrosis-related genes. This cluster represents about 20% of all human lesions but was found to be associated with severe cardiovascular events. A few mouse strains showed the highest correlations with two other human plaque types (e.g. BUB/Bnj for plaque type #1 and LG/J for plaque type #4), while the human plaque types #0 and #2 were much less reflected in analysed mouse models. Interestingly inflammatory pathways, important drivers in differentiating human clusters, only showed minor differences through all mice clusters. Conclusions Transcriptomic profiling of atherosclerotic lesions reveals that mice associate only partly with the human plaque phenotypes. The best associations were observed with those human plaques that correlate with a more severe clinical phenotype. The existence of different types of lesions observed in humans should be taken into account when choosing a mouse model to study atherosclerosis.

Comprehensive identification of gene expression fingerprints and biomarkers of sexual endocrine disruption in zebrafish embryo

Endocrine disruptors (EDs), capable of modulating the sex hormone system of an organism, can exert long-lasting negative effects on reproduction in both humans and the environment. For these reasons, the properties of EDs prevent a substance from being approved for marketing. However, regulatory testing to evaluate endocrine disruption is time-consuming, costly, and animal-intensive. Here, we combined sublethal zebrafish embryo assays with transcriptomics and proteomics for well-characterized endocrine disrupting reference compounds to identify predictive biomarkers for sexual endocrine disruption in this model. Using RNA and protein gene expression fingerprints from two different sublethal exposure concentrations, we identified specific signatures and impaired biological processes induced by ethinylestradiol, tamoxifen, methyltestosterone and flutamide 96 h post fertilization (hpf). Our study promotes vtg1 as well as cyp19a1b, fam20cl, lhb, lpin1, nr1d1, fbp1b, and agxtb as promising biomarker candidates for identifying and differentiating estrogen and androgen receptor agonism and antagonism. Evaluation of these biomarkers for pre-regulatory zebrafish embryo-based bioassays will help identify endocrine disrupting hazards of compounds at the molecular level. Such approaches additionally provide weight-of-evidence for the identification of putative EDs and may contribute significantly to a reduction in animal testing in higher tier studies.

Predicting cancer drug response using parallel heterogeneous graph convolutional networks with neighborhood interactions.

Due to cancer heterogeneity, the therapeutic effect may not be the same when a cohort of patients of the same cancer type receive the same treatment. The anticancer drug response prediction may help develop personalized therapy regimens to increase survival and reduce patients' expenses. Recently, graph neural network-based methods have aroused widespread interest and achieved impressive results on the drug response prediction task. However, most of them apply graph convolution to process cell line-drug bipartite graphs while ignoring the intrinsic differences between cell lines and drug nodes. Moreover, most of these methods aggregate node-wise neighbor features but fail to consider the element-wise interaction between cell lines and drugs. This work proposes a neighborhood interaction (NI)-based heterogeneous graph convolution network method, namely NIHGCN, for anticancer drug response prediction in an end-to-end way. Firstly, it constructs a heterogeneous network consisting of drugs, cell lines and the known drug response information. Cell line gene expression and drug molecular fingerprints are linearly transformed and input as node attributes into an interaction model. The interaction module consists of a parallel graph convolution network layer and a NI layer, which aggregates node-level features from their neighbors through graph convolution operation and considers the element-level of interactions with their neighbors in the NI layer. Finally, the drug response predictions are made by calculating the linear correlation coefficients of feature representations of cell lines and drugs. We have conducted extensive experiments to assess the effectiveness of our model on Cancer Drug Sensitivity Data (GDSC) and Cancer Cell Line Encyclopedia (CCLE) datasets. It has achieved the best performance compared with the state-of-the-art algorithms, especially in predicting drug responses for new cell lines, new drugs and targeted drugs. Furthermore, our model that was well trained on the GDSC dataset can be successfully applied to predict samples of PDX and TCGA, which verified the transferability of our model from cell line in vitro to the datasets in vivo. The source code can be obtained from https://github.com/weiba/NIHGCN. Supplementary data are available at Bioinformatics online.

Abstract PR013: Oct4 and Sox2 induce cellular transition of glioma stem cells to an immune suppressive, regulatory T cell-like state

Abstract Solid malignancies, including GBM, contain small subsets of cells that display stem-like properties (i.e. glioma stem cells or GSCs) and act as key determinants of therapeutic resistance and tumor recurrence. Mechanisms of GSC immune escape are considered fundamental to clinical GBM growth and recurrence. GBM cells escape antitumor immunity by modifying the tumor microenvironment by secreting immune-suppressive factors and recruiting anti-inflammatory/pro-oncogenic cells (e.g. T-regulatory cells, M2-like macrophages). However, whether subsets of GSCs can mimic T-reg-like functions by expressing proteins associated with T-reg immune-suppression remains unknown. Single-cell RNA sequencing analysis of GBM neurospheres revealed a previously unrecognized Oct4/Sox2high/FOXP3− cell subpopulation with high expression of TGFb1, CD39, CD73, PD-L1, and Galectin-1, a gene expression fingerprint typically associated with T-reg cells and their immune suppressive functions within the tumor microenvironment. Bioinformatics analysis of public databases shows that the above-mentioned genes are enriched in the mesenchymal GBM subtype and highly correlated with CD44 and TGFb type II receptor (TGFBR2) expression in both clinical GBM and primary GSCs. Mechanistically, Oct4 and Sox2 were found to directly induce TGFBR2 transcription concurrent with chromatin architectural changes (i.e. heterochromatin-to-euchromatin transition) involving the TGFBR2 gene. Transgenic TGFBR2 expression increased neurosphere cell growth capacity, upregulated expression of T-reg-like effector genes and enriched for CD44+ GSCs. Functionally, neurospheres expressing transgenic Oct4/Sox2 or transgenic TGFBR2 possess increased immunosuppressive capacity, and pharmacological inhibition of TGFBR2 depleted CD44+ GSCs and reduced immunosuppression in T-cell co-cultures. Taken together, we show that reprogramming events initiated by Oct4 and Sox2 induce a CD44+/FOX3P− GSC cell subset with a T-reg-like immunosuppressive transcriptional profile via a TGFBR2-dependent mechanism. This research provides the first description of such neoplastic cells in any malignancy and has high potential translational impact since targeting these tumor cell subsets and their immunosuppressive mechanisms may be critical to the successful development of GBM immunotherapies. Citation Format: Amanda Johnson, Jack Korleski, John Laterra, Hernando Lopez-Bertoni. Oct4 and Sox2 induce cellular transition of glioma stem cells to an immune suppressive, regulatory T cell-like state [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr PR013.

STEM-03. Oct4/Sox2 DRIVE AN IMMUNOSUPPRESSIVE GSC PHENOTYPE BY INDUCING T-REG EFFECTOR GENES VIA TGFBR2 SIGNALING

Abstract Solid malignancies, including GBM, contain small subsets of cells that display stem-like properties (i.e. glioma stem cells or GSCs) and act as key determinants of therapeutic resistance and tumor recurrence. Mechanisms of GSC immune escape are considered fundamental to clinical GBM growth and recurrence. GBM cells escape antitumor immunity by modifying the tumor microenvironment by secreting immune-suppressive factors and recruiting anti-inflammatory/pro-oncogenic cells (e.g. T-regulatory cells, M2-like macrophages). However, whether subsets of GSCs can mimic T-reg-like functions by expressing proteins associated with T-reg immune-suppression remains unknown. Single-cell RNA sequencing analysis of GBM neurospheres revealed a previously unrecognized Oct4/Sox2high/FOXP3- cell subpopulation with high expression of TGFb1, CD39, CD73, PD-L1, and Galectin-1, a gene expression fingerprint typically associated with T-reg cells and their immune suppressive functions within the tumor microenvironment. Bioinformatics analysis of public databases shows that the above-mentioned genes are enriched in the mesenchymal GBM subtype and highly correlated with CD44 and TGFb type II receptor (TGFBR2) expression in both clinical GBM and primary GSCs. Mechanistically, Oct4 and Sox2 were found to directly induce TGFBR2 transcription concurrent with chromatin architectural changes (i.e. heterochromatin-to-euchromatin transition) involving the TGFBR2 gene. Transgenic TGFBR2 expression increased neurosphere cell growth capacity, upregulated expression of T-reg-like effector genes and enriched for CD44+ GSCs. Functionally, neurospheres expressing transgenic Oct4/Sox2 or transgenic TGFBR2 possess increased immunosuppressive capacity, and pharmacological inhibition of TGFBR2 depleted CD44+ GSCs and reduced immunosuppression in Jurkat cell co-cultures. Taken together, we show that reprogramming events initiated by Oct4 and Sox2 induce a CD44+/FOX3P- GSC cell subset with a T-reg-like immunosuppressive transcriptional profile via a TGFBR2-dependent mechanism. This research provides the first description of such neoplastic cells in any malignancy and has high potential translational impact since targeting these tumor cell subsets and their immunosuppressive mechanisms may be critical to the successful development of GBM immunotherapies.

Toxicogenomic differentiation of functional responses to fipronil and imidacloprid in Daphnia magna.

Active substances of pesticides, biocides or pharmaceuticals can induce adverse side effects in the aquatic ecosystem, necessitating environmental hazard and risk assessment prior to substance registration. The freshwater crustacean Daphnia magna is a model organism for acute and chronic toxicity assessment representing aquatic invertebrates. However, standardized tests involving daphnia are restricted to the endpoints immobility and reproduction and thus provide only limited insights into the underlying modes-of-action. Here, we applied transcriptome profiling to a modified D. magna Acute Immobilization test to analyze and compare gene expression profiles induced by the GABA-gated chloride channel blocker fipronil and the nicotinic acetylcholine receptor (nAChR) agonist imidacloprid. Daphnids were expose to two low effect concentrations of each substance followed by RNA sequencing and functional classification of affected gene ontologies and pathways. For both insecticides, we observed a concentration-dependent increase in the number of differentially expressed genes, whose expression changes were highly significantly positively correlated when comparing both test concentrations. These gene expression fingerprints showed virtually no overlap between the test substances and they related well to previous data of diazepam and carbaryl, two substances targeting similar molecular key events. While, based on our results, fipronil predominantly interfered with molecular functions involved in ATPase-coupled transmembrane transport and transcription regulation, imidacloprid primarily affected oxidase and oxidoreductase activity. These findings provide evidence that systems biology approaches can be utilized to identify and differentiate modes-of-action of chemical stressors in D. magna as an invertebrate aquatic non-target organism. The mechanistic knowledge extracted from such data will in future contribute to the development of Adverse Outcome Pathways (AOPs) for read-across and prediction of population effects.

Dysregulation of Components of the Inflammasome Machinery After Bariatric Surgery: Novel Targets for a Chronic Disease.

Obesity is a metabolic chronic disease with important associated morbidities and mortality. Bariatric surgery is the most effective treatment for maintaining long-term weight loss in severe obesity and, consequently, for decreasing obesity-related complications, including chronic inflammation. To explore changes in components of the inflammasome machinery after bariatric surgery and their relation with clinical/biochemical parameters at baseline and 6 months after bariatric surgery. Twenty-two patients with morbid-obesity that underwent bariatric surgery (sleeve gastrectomy and Roux-en-Y gastric bypass) were included. Epidemiological/clinical/anthropometric/biochemical evaluation was performed at baseline and 6 months after bariatric surgery. Inflammasome components and inflammatory-associated factors [nucleotide-binding oligomerization domain-like receptors (NLRs), inflammasome activation components, cytokines and inflammation/apoptosis-related components, and cell-cycle and DNA-damage regulators) were evaluated in peripheral blood mononuclear cells (PBMCs) at baseline and 6 months after bariatric surgery. Clinical molecular correlations/associations were analyzed. Functional parameters (lipid accumulation/viability/apoptosis) were analyzed in response to specific inflammasome components silencing in liver HepG2 cells). A profound dysregulation of inflammasome components after bariatric surgery was found, especially in NLRs and cell-cycle and DNA damage regulators. Several components were associated with baseline metabolic comorbidities including type 2 diabetes (C-C motif chemokine ligand 2/C-X-C motif chemokine receptor 1/sirtuin 1), hypertension (absent in melanoma 2/ASC/purinergic receptor P2X 7), and dyslipidemia [C-X-C motif chemokine ligand 3 (CXCL3)/NLR family pyrin domain containing (NLRP) 7) and displayed changes in their molecular profile 6 months after bariatric surgery. The gene expression fingerprint of certain factors NLR family CARD domain containing 4 (NLRC4)/NLRP12/CXCL3)/C-C motif chemokine ligand 8/toll-like receptor 4) accurately differentiated pre- and postoperative PBMCs. Most changes were independent of the performed surgical technique. Silencing of NLRC4/NLRP12 resulted in altered lipid accumulation, apoptosis rate, and cell viability in HepG2 cells. Bariatric surgery induces a profound alteration in the gene expression pattern of components of the inflammasome machinery in PBMCs. Expression and changes of certain inflammasome components are associated to baseline metabolic comorbidities, including type 2 diabetes, and may be related to the improvement and reversion of some obesity-related comorbidities after bariatric surgery.

A pan-cancer transcriptomic study showing tumor specific alterations in central metabolism

Recently, there has been a resurgence of interest in metabolic rewiring of tumors to identify clinically relevant genes. However, most of these studies have had either focused on individual tumors, or are too general, providing a broad outlook on overall changes. In this study, we have first curated an extensive list of genes encoding metabolic enzymes and metabolite transporters relevant to carbohydrate, fatty acid and amino acid oxidation and biosynthesis. Next, we have used publicly available transcriptomic data for 20 different tumor types from The Cancer Genome Atlas Network (TCGA) and focused on differential expression of these genes between tumor and adjacent normal tissue. Our study revealed major transcriptional alterations in genes that are involved in central metabolism. Most tumors exhibit upregulation in carbohydrate and amino acid transporters, increased glycolysis and pentose phosphate pathway, and decreased fatty acid and amino acid oxidation. On the other hand, the expression of genes of the tricarboxylic acid cycle, anaplerotic reactions and electron transport chain differed between tumors. Although most transcriptomic alterations were conserved across many tumor types suggesting the initiation of common regulatory programs, expression changes unique to specific tumors were also identified, which can provide gene expression fingerprints as potential biomarkers or drug targets. Our study also emphasizes the value of transcriptomic data in the deeper understanding of metabolic changes in diseases.

Altered Expression of Ion Channels in White Matter Lesions of Progressive Multiple Sclerosis: What Do We Know About Their Function?

Despite significant advances in our understanding of the pathophysiology of multiple sclerosis (MS), knowledge about contribution of individual ion channels to axonal impairment and remyelination failure in progressive MS remains incomplete. Ion channel families play a fundamental role in maintaining white matter (WM) integrity and in regulating WM activities in axons, interstitial neurons, glia, and vascular cells. Recently, transcriptomic studies have considerably increased insight into the gene expression changes that occur in diverse WM lesions and the gene expression fingerprint of specific WM cells associated with secondary progressive MS. Here, we review the ion channel genes encoding K+, Ca2+, Na+, and Cl− channels; ryanodine receptors; TRP channels; and others that are significantly and uniquely dysregulated in active, chronic active, inactive, remyelinating WM lesions, and normal-appearing WM of secondary progressive MS brain, based on recently published bulk and single-nuclei RNA-sequencing datasets. We discuss the current state of knowledge about the corresponding ion channels and their implication in the MS brain or in experimental models of MS. This comprehensive review suggests that the intense upregulation of voltage-gated Na+ channel genes in WM lesions with ongoing tissue damage may reflect the imbalance of Na+ homeostasis that is observed in progressive MS brain, while the upregulation of a large number of voltage-gated K+ channel genes may be linked to a protective response to limit neuronal excitability. In addition, the altered chloride homeostasis, revealed by the significant downregulation of voltage-gated Cl− channels in MS lesions, may contribute to an altered inhibitory neurotransmission and increased excitability.

Langerhans cells rely on good neighbors to overcome gene deficiencies.

Abstract We recently identified that epidermal resident Langerhans Cells (LCs) acquire gene expression fingerprints from surrounding Keratinocytes (KCs) in the form of mRNA and protein. In the present study, we aimed to determine whether this transfer can also overcome gene deficiencies. For this purpose, using the Cre/Lox system, we specifically deleted genes for Connexin 43 (Cx43), MyD88, and MHC-II in LCs. While all three genes underwent recombination, reduced protein levels were only observed for MHC-II, whereas Cx43 and MyD88 protein levels remained unaltered. Considering that KCs lack MHC-II, but express Cx43 and MyD88 at high levels, we posit that LCs can acquire gene products from surrounding KCs to overcome their own deficiencies if those products are available. In summary, we present evidence that cells can compensate for gene deficiencies if the surrounding cells can provide. These findings highlight the limitations of cell-specific gene deletion, and could provide an explanation as to why certain gene deletions do not lead to measurable deficiencies.

Toxicogenomic fin(ger)prints for thyroid disruption AOP refinement and biomarker identification in zebrafish embryos

Endocrine disruption (ED) can trigger far-reaching effects on environmental populations, justifying a refusal of market approval for chemicals with ED properties. For the hazard assessment of ED effects on the thyroid system, regulatory decisions mostly rely on amphibian studies. Here, we used transcriptomics and proteomics for identifying molecular signatures of interference with thyroid hormone signaling preceding physiological effects in zebrafish embryos. For this, we analyzed the thyroid hormone 3,3′,5-triiodothyronine (T3) and the thyroid peroxidase inhibitor 6-propyl-2-thiouracil (6-PTU) as model substances for increased and repressed thyroid hormone signaling in a modified zebrafish embryo toxicity test. We identified consistent gene expression fingerprints for both modes-of-action (MoA) at sublethal test concentrations. T3 and 6-PTU both significantly target the expression of genes involved in muscle contraction and functioning in an opposing fashion, allowing for a mechanistic refinement of key event relationships in thyroid-related adverse outcome pathways in fish. Furthermore, our fingerprints identify biomarker candidates for thyroid disruption hazard screening approaches. Perspectively, our findings will promote the AOP-based development of in vitro assays for thyroidal ED assessment, which in the long term will contribute to a reduction of regulatory animal tests.

Cardioembolic Ischemic Stroke Gene Expression Fingerprint in Blood: a Systematic Review and Verification Analysis.

An accurate etiological classification is key to optimize secondary prevention after ischemic stroke, but the cause remains undetermined in one third of patients. Several studies pointed out the usefulness of circulating gene expression markers to discriminate cardioembolic (CE) strokes, mainly due to atrial fibrillation (AF), while only exploring them in small cohorts. A systematic review of studies analyzing high-throughput gene expression in blood samples to discriminate CE strokes was performed. Significantly dysregulated genes were considered as candidates, and a selection of them was validated by RT-qPCR in 100 patients with defined CE or atherothrombotic (LAA) stroke etiology. Longitudinal performance was evaluated in 12 patients at three time points. Their usefulness as biomarkers for AF was tested in 120 cryptogenic strokes and 100 individuals at high-risk for stroke. Three published studies plus three unpublished datasets were considered for candidate selection. Sixty-seven genes were found dysregulated in CE strokes. CREM, PELI1, and ZAK were verified to be up-regulated in CE vs LAA (p = 0.010, p = 0.003, p < 0.001, respectively), without changes in their expression within the first 24h after stroke onset. The combined up-regulation of these three biomarkers increased the probability of suffering from CE stroke by 23-fold. In cryptogenic strokes with subsequent AF detection, PELI1 and CREM showed overexpression (p = 0.017, p = 0.059, respectively), whereas in high-risk asymptomatic populations, all three genes showed potential to detect AF (p = 0.007, p = 0.007, p = 0.015). The proved discriminatory capacity of these gene expression markers to detect cardioembolism even in cryptogenic strokes and asymptomatic high-risk populations might bring up their use as biomarkers.

Keratinocytes Share Gene Expression Fingerprint with Epidermal Langerhans Cells via mRNA Transfer

The immune functions of epithelia-resident dendritic cells are influenced by epithelial-derived cytokines. Here we identified a communication form between tissue-resident dendritic cells and niche cells that allows direct intracellular material exchange between the parties. We show that many keratinocyte (KC)-specific molecules such as keratins and adhesion molecules could be detected in the epidermal-resident Langerhans cells (LCs) as mRNA and protein. Furthermore, KC-derived Cre led to genetic recombination in the LCs. We also found that LCs containing KC-derived material were more prone to migration. The KC-specific signatures were transferred from KCs to LCs through an exosome-independent mechanism that likely involved nanotubes/dendrites. The transfer of material between epithelial cells and epithelia-associated dendritic cells was not limited to mice or to KC-to-LC transfer. Taken together, these data suggest that the epithelial environment might have a long-term effect on dendritic cell biology and that genetic tools that specifically target epithelial cells also affect tissue-resident immune cells.

Antioxidant, gene expression and metabolomics fingerprint analysis of Arabidopsis thaliana treated by foliar spraying of ZnSe quantum dots and their growth inhibition of Agrobacterium tumefaciens.

Nanotechnology, new fascinating field of science, is bringing many application's options. However, it is necessary to understand their potential environmental risk and toxicity. Zinc selenide quantum dots (ZnSe QDs) are getting valuable due to wide industrial usage, mainly as cadmium free diodes or stabilizing ligand. Thanks to unique properties, they could also open the possibilities of application in the agriculture. Their effects on living organisms, including plants, are still unknown. Therefore, the attention of this work was given to antioxidant response of Arabidopsis thaliana to 100 and 250 μM ZnSe QDs foliar feeding. ZnSe QDs treatment had no statistically significant differences in morphology but led to increased antioxidant response in the leaves at the level of gene expression and production secondary antioxidant metabolites. Concurrently, analysis of growth properties of Agrobacterium tumefaciens was done. 250 μM ZnSe solution inhibited the Agrobacterium tumefaciens viability by 60%. This is the first mention about effect ZnSe QDs on the plants. Although QDs induced oxidative stress, the apply treatment dose of ZnSe QDs did not have significant toxic effect on the plants and even no morphological changes were observed. However, the same amount of ZnSe QD induced an inhibitory effect on Agrobacterium tumefaciens.

PO-046 Gene expression profiling of peripheral blood mononuclear cells in young male trampoline athletes

Objective Evidence indicates that physical activity influence bone health. However, the molecular mechanisms mediating the beneficial adaptations to exercise are not well understood. The purpose of this study was to examine the differentially expressed genes in PBMC between athletes and healthy controls, and to analyze the important functional genes and signal pathways that cause increased bone mineral density in athletes, in order to further reveal the molecular mechanisms of exercise promoting bone health.&#x0D; Methods Five professional trampoline athletes and five age-matched untrained college students participated in this study. Used the human expression Microarray V4.0 expression profiling chip to detect differentially expressed genes in the two groups, and performed KEGG Pathway analysis and application of STRING database to construct protein interaction Network; Real-Time PCR technology was used to verify the expression of some differential genes. &#x0D; Results Compared with healthy controls, there were significant improvement in lumbar spine bone mineral density, and 236 up-regulated as well as 265 down-regulated in serum samples of athletes. The differentially expressed genes involved 28 signal pathways, such as cell adhesion molecules. Protein interaction network showed that MYC was at the core node position. Real-time PCR results showed that the expression levels of CD40 and ITGα6 genes in the athletes were up-regulated compared with the healthy controls, the detection results were consistent with that of the gene chip.&#x0D; Conclusions The findings highlight that long-term high-intensity trampoline training could induce transcriptional changes in PBMC of the athletes. These data suggest that gene expression fingerprints can serve as a powerful research tool to design novel strategies for monitoring exercise. The findings of the study also provide support for the notion that PBMC could be used as a substitute to study exercise training that affects bone health.

Hippo signaling in the ovary and polycystic ovarian syndrome.

To provide a commentary on our understanding of the role that the Hippo signaling pathway may play in patients with polycystic ovarian syndrome (PCOS) and how this understanding may impact the diagnosis of PCOS. We assessed publications discussing the role of the Hippo signaling pathway in the ovary. In particular, we discuss how Hippo signaling disruption after ovarian fragmentation, combined with treating ovarian fragments with phosphatase and tensin homolog (PTEN) inhibitors and phosphoinositide-3-kinase stimulators to augment AKT signaling, has been used in treatment of patients with primary ovarian insufficiency. Furthermore, we discuss our own data on variations in Hippo signaling pathway gene expression in cumulus cells isolated from women undergoing IVF with a previous diagnosis of PCOS. Aberrant Hippo signaling in PCOS patients is likely a contributing mechanism to the multifactorial etiology of the disease. Given the challenge of discerning the underlying etiology of oligo-ovulation in some patients, especially those with normal body mass indices, and the need for customized stimulation protocols for PCOS patients who have an increased risk of over-response and higher percentage of immature oocyte yield, it is important to identify these patients prior to treatment. Hippo gene expression fingerprints could potentially be used to more accurately define patients with PCOS. Additionally, targeting this pathway with pharmacologic agents could lead to non-surgical therapeutic options for PCOS.

Absence of NEFL in patient-specific neurons in early-onset Charcot-Marie-Tooth neuropathy.

ObjectiveWe used patient-specific neuronal cultures to characterize the molecular genetic mechanism of recessive nonsense mutations in neurofilament light (NEFL) underlying early-onset Charcot-Marie-Tooth (CMT) disease.MethodsMotor neurons were differentiated from induced pluripotent stem cells of a patient with early-onset CMT carrying a novel homozygous nonsense mutation in NEFL. Quantitative PCR, protein analytics, immunocytochemistry, electron microscopy, and single-cell transcriptomics were used to investigate patient and control neurons.ResultsWe show that the recessive nonsense mutation causes a nearly total loss of NEFL messenger RNA (mRNA), leading to the complete absence of NEFL protein in patient's cultured neurons. Yet the cultured neurons were able to differentiate and form neuronal networks and neurofilaments. Single-neuron gene expression fingerprinting pinpointed NEFL as the most downregulated gene in the patient neurons and provided data of intermediate filament transcript abundancy and dynamics in cultured neurons. Blocking of nonsense-mediated decay partially rescued the loss of NEFL mRNA.ConclusionsThe strict neuronal specificity of neurofilament has hindered the mechanistic studies of recessive NEFL nonsense mutations. Here, we show that such mutation leads to the absence of NEFL, causing childhood-onset neuropathy through a loss-of-function mechanism. We propose that the neurofilament accumulation, a common feature of many neurodegenerative diseases, mimics the absence of NEFL seen in recessive CMT if aggregation prevents the proper localization of wild-type NEFL in neurons. Our results suggest that the removal of NEFL as a proposed treatment option is harmful in humans.