Fold Change In Expression Research Articles

Exploring Flowering Genes in Isabgol (Plantago ovata Forsk.) Through Transcriptome Analysis

Flowering is one of the major developmental processes that govern the economic yield of crop plants. However, little is known about the molecular mechanisms underlying flowering in Isabgol, an important high-value medicinal crop. Here, we analyzed the leaf transcriptome of early and late flowering genotypes by high throughput next-generation sequencing to uncover the genes and pathways involved in flowering time and flower development. Illumina paired-end sequencing of Isabgol leaves at the stem elongation stage, generated 8,976,119 and 4,282,684 reads respectively in DPO-14 (early flowering) and DPO-185 (late flowering) genotypes. The sequence assembly resulted in 40,175 and 39,533 transcripts respectively in early and late genotypes. A total of 17,768 (95.50) in DPO-14 and 21,255 (94.10) in DPO-185 CDS were annotated. There were 8981CDS were differentially expressed of which 1220 (13.58%) were significantly upregulated while 1485 (16.53%) CDS were significantly downregulated in DPO-185 compared with DPO-14. In total, 229 genes were identified belongs to distinct flowering pathways in Isabgol. A putative schematic network of flowering pathway regulation in Isabgol was proposed. Significant DEGs (60 genes) related to flowering time and flower development were detected between the early and late flowering genotypes. Significant differences in fold change expression of 17 genes were observed in early and late flowering genotypes. Many differentially expressed genes (DEGs) involved in flowering time and flower development were identified. The expression data will serve as a resource for unraveling the functions of specific genes involved in flower development in Isabgol and other plants. These findings are significant for further understanding of the molecular basis for flowering time regulation, breeding, and molecular biology in Isabgol as well as other crop plants.

Pilot study on cardiogenic differentiation capability of rabbit mesenchymal stem cells

Cardiovascular diseases are still one of the most common reasons for mortality in humans. Mesenchymal stem cells (MSCs) are preferable in cardiac regeneration cell-based therapies because of their allogeneic and high proliferative potential. The electrophysiological properties of the rabbit heard is closer to human than the mouse. The current study aimed to trace mRNA expression changes of two stemness/cardiogenic differentiation ability-related transcriptionala factors OCT4 and GATA4 in rabbit MSCs during early stages of induced cardiomyocyte differentiation in vitro. The mesenchymal stem cell originated from different anatomical areas-subcutaneous, visceral, bone marrow and pericardial tissue. The cardiac differentiation protocol for mouse embryonic stem cells in hanging drop was adopted in rabbit MSCs. The best formed embryonal bodies (EBs) like structures were collected and cultivated on gelatin-coated plates. The total mRNA was obtained before cardiac differentiation and on the 6th day after it. SYBER based real-time PCR was performed to evaluate the mRNA expression fold-changes of OCT4 and GATA4. The cultivation of MSCs in hanging drops during cardiac differentiation induced EBs formation, without any contractile activity up to the 6th day of the differentiation in all cell types. The applied differentiation protocol significantly downregulated GATA4 expression in ADSCs - EBs, while in BMSCs, both target genes were significantly upregulated. In conclusion, the adopted cardiac differentiation protocol from mouse embryonic stem cells could be a useful approach for rabbit bone marrow mesenchymal stem cells. Since the rest of the cells revealed weak cardiogenic capability at this early stage, some modifications of induction protocols should be considered.

The diagnostic value of miR-92a, -143, and -145 expression levels in patients with colorectal adenocarcinoma from Romania.

MicroRNAs (miRNAs) refers to a small, short non-coding RNA of endogenous class. They have shown to have an increasingly altered expression in many types of cancer, including colorectal cancer (CRC).In the present study, miRNA TaqManMGB and qRT-PCR was used to quantify the expression and clinical significance of 3 mature human miRNA in 82 pairs of colorectal adenocarcinoma tissues and normal adjacent tissue samples (NATS) collected from patients of the south-east part of Romania. Differences between CRC and NATS were analyzed using Wilcoxon test, while correlations between miRNAs expression levels and clinicopathological features were examined using non-parametric tests. In addition, the ability of selected miRNAs to function as biomarkers and, as potential indicators in CRC prognosis was also examined.When the miRNA expression was compared in CRC related NATS, miR-143, and miR-145 were significantly underexpressed (4.99 ± –1.02 vs –5.66 ± –1.66, P < .001; –4.85 ± –0.59 vs –9.27 ± –1.51, P < .001, respectively), while the pattern of miR-92a was significantly overexpressed (–5.55 ± –2.83 vs –4.92 ± –2.44, P < .001). Moreover, the expression levels of selected miRNAs were identified to be correlated with gradual increases in fold change expression with the depth of tumor invasion, lymph node invasion, and maximal increases with distant metastasis. Furthermore, the receiver operating characteristic analysis demonstrated that potential diagnostic of miR-143, miR-145, and miR-92a in discriminating CRC from NATS, with the area under the curve of 0.74, 0.85, and 0.84 respectively. The Kaplan–Meier and the log-rank test showed that a high level of miR-92a and low levels of miR-143 and miR-145 predicted poor survival rate in our cohorts.In conclusion, we can summarize that miR-145 and miR-143 are decreased, while miR-92 is increased in CRC compared to NATS, and associated with different stages of CRC pathogenesis. Thus, the expression of selected miRNAs can represent potential diagnostic and prognostic tools in patients with CRC from Romania.

Relationship between Chromosomal Aberrations and Gene Expressions in the p53 Pathway in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a neoplasm characterized by excessive accumulation of B lymphocytes in the peripheral blood, bone marrow and lymph nodes. We assessed the expressions of 22 genes in the p53 pathway in 30 CLL patients and 15 healthy subjects by a RT2 Profiler PCR (polymerase chain reaction) Array technique and their relation to cytogenetic aberrations detected by fluorescent in situ hybridization (FISH). Our Student’s t-test results indicated that ATM, ATR, BAX, CASP9, CDK4, CDKN2A, CHEK1, CHEK2, E2F3, MCL1, MDM2, MDM4, PCNA, RB1, P53 and BCL2 genes were statistically significant (p <0.001). For six genes (APAF1, CDKN1A, E2F1, GADD45A, PTEN and PTX3) were not statistically significant. The ATM, ATR, BAX, CASP9, CDK4, CDKN1A, CDKN2A, CHEK1, CHEK2, MDM2, MDM4, PCNA, RB1, P53, E2F1, GADD45A and BCL2 genes were found to be upregulated by the 2-ᐃᐃCt (relative fold change in gene expression) method. The highest up-regulation was detected in CDKN2A and BCL2 genes, 10.22- and 8.51-fold, respectively. On the other hand, the PTX3 gene with a fold regulation of 1.84 was found to the highest downregulation. Overall, the CDNK2A BCL2 and PTX3 genes are related to the mechanism of the disease in the p53 pathway and may be an important predictor of the prognosis of the disease. The BCL2 gene may be associated with increased risk of developing CLL. We suggest that the PTX3 gene may be considered as a marker associated with CLL disease. The CDKN2A gene expression seems to play a protective role in CLL.

Abstract 1158: Hormone gene expression and mammographic breast density in postmenopausal women

Abstract Introduction: Mammographic breast density (MBD), which reflects the amount of epithelial and stromal tissues relative to adipose tissue in the breast, is a strong risk factor for breast cancer. MBD declines with menopause, highlighting the role of endogenous hormones. Nevertheless, studies evaluating the associations of circulating hormone levels with MBD report conflicting results. Only a limited number of studies have addressed the genomic signatures of MBD, which may better capture the broader transcriptional changes associated with MBD and more accurately quantify hormonal exposure than circulating hormone levels. We therefore investigated the associations of hormonal gene expression with MBD in postmenopausal women. Methods: We recruited 368 mid-life postmenopausal women (ages 50-64 years) who had annual screening mammogram at Washington University School of Medicine, St. Louis, MO between October 2017 and September 2018. Each participant completed a blood draw and a questionnaire on breast cancer risk factors. We used Volpara software to measure volumetric percent density (VPD, %), dense volume (VD, cm3) and non-dense volume (NDV, cm3). Prolactin (PRL), progesterone (PGR), estrogen receptor 1 (ESR1), signal transducer and activator of transcription (STAT1), and STAT5 gene expression were measured in total RNA isolated from plasma using NanoString nCounter system. Statistical analyses were performed with the NanoString nSolver Analysis Software. Fold changes in gene expression and p-values were calculated using nCounter default settings. Analyses were adjusted for body mass index (BMI), age, age at menarche, parity/age at first birth, race, and menopausal hormone use. VPD, DV, and NDV were log10 transformed to ensure the normality of the residuals. Results: The mean age of study participants was 57.9 years. The mean BMI was 31.3kg/m2. The average VPD, DV, and NDV were 6.2%, 121.2 cm3, and 2134.4 cm3, respectively. There were strong correlations between STAT1 and STAT5 gene expression (r=0.59, p-value&amp;lt;0.0001), and moderate correlations between ESR1 and PGR gene expression (r=0.43, p-value&amp;lt;0.0001). ESR1 gene expression was associated with VPD, while PGR and STAT5 were marginally associated with NDV. One unit increase in ESR1 gene expression was associated with a 29% (95% CI 2-64%) increase in VPD (p-value=0.03). One unit increase in STAT5 gene expression was associated with an 8% (p-value=0.05) increase in NDV, while one unit increase in PGR gene expression was associated with a 20% (p-value=0.06) decrease in NDV. Hormone gene expression was not associated with DV. Conclusions: We provide novel data on the associations of endogenous hormone gene expression with MBD in postmenopausal women. Future studies characterizing transcriptomic changes in hormone signaling and MBD are needed, as targeting these pathways could be used to reduce MBD for breast cancer prevention. Citation Format: Rachel L. Mintz, Mei Wang, Shuai Xu, Chris Markovic, Adetunji T. Toriola. Hormone gene expression and mammographic breast density in postmenopausal women [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1158.

Multiomics of World Trade Center Particulate Matter-induced Persistent Airway Hyperreactivity. Role of Receptor for Advanced Glycation End Products.

Pulmonary disease after World Trade Center particulate matter (WTC-PM) exposure is associated with dyslipidemia and the receptor for advanced glycation end products (RAGE); however, the mechanisms are not well understood. We used a murine model and a multiomics assessment to understand the role of RAGE in the pulmonary long-term effects of a single high-intensity exposure to WTC-PM. After 1 month, WTC-PM-exposed wild-type (WT) mice had airway hyperreactivity, whereas RAGE-deficient (Ager-/-) mice were protected. PM-exposed WT mice also had histologic evidence of airspace disease, whereas Ager-/- mice remained unchanged. Inflammatory mediators such as G-CSF (granulocyte colony-stimulating factor), IP-10 (IFN-γ-induced protein 10), and KC (keratinocyte chemoattractant) were differentially expressed after WTC-PM exposure. WTC-PM induced α-SMA, DIAPH1 (protein diaphanous homolog 1), RAGE, and significant lung collagen deposition in WT compared with Ager-/- mice. Compared with WT mice with PM exposure, relative expression of phosphorylated to total CREB (cAMP response element-binding protein) and JNK (c-Jun N-terminal kinase) was significantly increased in the lung of PM-exposed Ager-/- mice, whereas Akt (protein kinase B) was decreased. Random forests of the refined lung metabolomic profile classified subjects with 92% accuracy; principal component analysis captured 86.7% of the variance in three components and demonstrated prominent subpathway involvement, including known mediators of lung disease such as vitamin B6 metabolites, sphingolipids, fatty acids, and phosphatidylcholines. Treatment with a partial RAGE antagonist, pioglitazone, yielded similar fold-change expression of metabolites (N6-carboxymethyllysine, 1-methylnicotinamide, N1+N8-acetylspermidine, and succinylcarnitine [C4-DC]) between WT and Ager-/- mice exposed to WTC-PM. RAGE can mediate WTC-PM-induced airway hyperreactivity and warrants further investigation.

Meta-QTL analysis and identification of candidate genes for drought tolerance in bread wheat (Triticum aestivum L.).

Meta-QTL (MQTL) analysis for drought tolerance was undertaken in bread wheat to identify consensus and robust MQTLs using 340 known QTLs from 11 earlier studies; 13 MQTLs located on 6 chromosomes (1D, 3B, 5A, 6D, 7A and 7D) were identified, with maximum of 4 MQTLs on chromosome 5A. Mean confidence intervals for MQTLs were much narrower (mean, 6.01cM; range 2.07-19.46cM), relative to those in original QTLs (mean, 13.6cM; range, 1.0-119.1cM). Two MQTLs, namely MQTL4 and MQTL12, were major MQTLs with potential for use in marker-assisting breeding. As many as 228 candidate genes (CGs) were also identified using 6 of the 13 MQTLs. In-silico expression analysis of these 228 CGs allowed identification of 14 important CGs, with + 3 to - 8 fold change in expression under drought (relative to normal conditions) in a tolerant cv. named TAM107. These CGs encoded proteins belonging to the following families: NAD-dependent epimerase/dehydratase, protein kinase, NAD(P)-binding domain protein, heat shock protein 70 (Hsp70), glycosyltransferase 2-like, etc. Important MQTLs and CGs identified in the present study should prove useful for future molecular breeding and for thestudy of molecular basis of drought tolerance in cereals in general and wheat in particular.

DEBay: A computational tool for deconvolution of quantitative PCR data for estimation of cell type-specific gene expression in a mixed population

The expression of a gene is commonly estimated by quantitative PCR (qPCR) using RNA isolated from a large number of pooled cells. Such pooled samples often have subpopulations of cells with different levels of expression of the target gene. Estimation of gene expression from an ensemble of cells obscures the pattern of expression in different subpopulations. Physical separation of various subpopulations is a demanding task. We have developed a computational tool, Deconvolution of Ensemble through Bayes-approach (DEBay), to estimate cell type-specific gene expression from qPCR data of a mixed population. DEBay estimates Normalized Gene Expression Coefficient (NGEC), which is a relative measure of the expression of the target gene in each cell type in a population. NGEC has a direct algebraic correspondence with the normalized fold change in gene expression measured by qPCR. DEBay can deconvolute both time-dependent and -independent gene expression profiles. It uses the Bayesian method of model selection and parameter estimation. We have evaluated DEBay using synthetic and real experimental data. DEBay is implemented in Python. A GUI of DEBay and its source code are available for download at SourceForge (https://sourceforge.net/projects/debay).

Mechanistic studies of cytotoxic activity of the mesoionic compound MIH 2.4Bl in MCF-7 breast cancer cells.

In the present study, the cytotoxic effects of a 1,3-thiazolium-5-thiolate derivative of a mesoionic compound, MIH 2.4Bl, were assessed in the MCF-7 breast cancer cell line. The cytotoxic effects of MIH 2.4Bl were determined using a crystal violet assay. Using a dose-response curve, the IC50 value of MIH 2.4Bl was determined to be 45.8±0.8 µM. Additionally, the effects of MIH 2.4Bl on mitochondrial respiration were characterized using oxygen consumption rate analysis. Treating MCF-7 cells with increasing concentrations of MIH 2.4Bl resulted in a significant reduction in all mitochondrial respiratory parameters compared with the control cells, indicative of an overall decrease in mitochondrial membrane potential. The induction of autophagy by MIH 2.4Bl was also examined by measuring changes in the expression of protein markers of autophagy. As shown by western blot analysis, treatment of MCF-7 cells with MIH 2.4Bl resulted in increased protein expression levels of Beclin-1 and ATG5, as well as an increase in the microtubule-associated protein 1A/1B light chain 3B (LC3B)-II to LC3B-I ratio compared with the control cells. Microarray analysis of changes in gene expression following MIH 2.4Bl treatment demonstrated 3,659 genes exhibited a fold-change ≥2. Among these genes, 779 were up-regulated, and 2,880 were down-regulated in cells treated with MIH 2.4Bl compared with the control cells. Based on the identity of the transcripts and fold-change of expression, six genes were selected for verification by reverse transcription-quantitative (RT-q)PCR; activating transcription factor 3, acidic repeat-containing protein, heparin-binding EGF-like growth factor, regulator of G-protein signaling 2, Dickkopf WNT signaling pathway inhibitor 1 and adhesion molecule with Ig like domain 2. The results of RT-qPCR analysis of RNA isolated from control and MIH 2.4Bl treated cells were consistent with the expression changes identified by microarray analysis. Together, these results suggest that MIH 2.4Bl may be a promising candidate for treating breast cancer and warrants further in vitro and in vivo investigation.

Molecular characterisation of TP53 mutated squamous cell carcinomas of the lung to identify putative targets for therapy.

Personalised cancer treatment depends on identification of therapeutically relevant biological subgroups of patients for assessing effect of treatment and to discover new therapeutic options. By analyses in heterogeneous patient populations, the effects may be lost in noise. Squamous cell carcinoma of the lung is a major killer worldwide. Despite recent advances, mortality is high and response to therapies varies greatly from patient to patient. Target search in biologically relevant subgroups may identify treatment options not so far discovered. A total of 198 patients undergoing surgery for squamous cell carcinomas of the lung were included in the study. The tumours were analysed for copy number alterations (n = 152) and gene expression from tumour (n = 188) and normal lung (n = 21), with both data levels present in 140 patients. We studied alterations in tumours harbouring mutations in TP53 and in previously published gene expression subtypes. Genes with consistent alterations in both genomic levels were identified as putative biomarkers. Results were validated in TCGA. The most convincing biomarker in TP53 mutated squamous cell carcinomas of the lung was BIRC5 with amplification in 36% of mutated samples, 5% in wild‐type samples and a 17%‐fold change of expression between TP53 mutated tumours and normal lung tissue. BIRC5 was significantly altered in the classical and primitive subtypes. We suggest BIRC5 as a putative predictive biomarker and putative druggable target in squamous cell lung carcinomas harbouring TP53 mutation or classified as classical and primitive subtypes.

Surface Modifications of an Organic Polymer-Based Microwire Platform for Sustained Release of an Anti-Inflammatory Drug.

Brain machine interfaces (BMIs), introduced into the daily lives of individuals with injuries or disorders of the nervous system such as spinal cord injury, stroke, or amyotrophic lateral sclerosis, can improve the quality of life. BMIs rely on the capability of microelectrode arrays to monitor the activity of large populations of neurons. However, maintaining a stable, chronic electrode-tissue interface that can record neuronal activity with a high signal-to-noise ratio is a key challenge that has limited the translation of such technologies. An electrode implant injury leads to a chronic foreign body response that is well-characterized and shown to affect the electrode-tissue interface stability. Several strategies have been applied to modulate the immune response, including the application of immunomodulatory drugs applied both systemically and locally. While the use of passive drug release at the site of injury has been exploited to minimize neuroinflammation, this strategy has all but failed as a bolus of anti-inflammatory drugs is released at predetermined times that are often inconsistent with the ongoing innate inflammatory process. Common strategies do not focus on the proper anchorage of soft hydrogel scaffolds on electrode surfaces, which often results in delamination of the porous network from electrodes. In this study, we developed a microwire platform that features a robust yet soft biocompatible hydrogel coating, enabling long-lasting drug release via formation of drug aggregates and dismantlement of hydrophilic biodegradable three-dimensional polymer networks. Facile surface chemistry is developed to functionalize polyimide-coated electrodes with the covalently anchored porous hydrogel network bearing large numbers of highly biodegradable ester groups. Exponential long-lasting drug release is achieved using such hydrogels. We show that the initial state of dexamethasone (Dex) used to formulate the hydrogel precursor solution plays a cardinal role in engineering hydrophilic networks that enable a sustained and long-lasting release of the anti-inflammatory agent. Furthermore, utilization of a high loading ratio that exceeds the solubility of Dex leads to the encapsulation of Dex aggregates that regulate the release of this anti-inflammatory agent. To validate the anti-inflammatory effect of the hydrogel-functionalized Dex-loaded microwires, an in vivo preliminary study was performed in adult male rats (n = 10) for the acute time points of 48 h and 7 days post implant. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess the mRNA expression of certain inflammatory-related genes. In general, a decrease in fold-change expression was observed for all genes tested for Dex-loaded wires compared with controls (functionalized but no drug). The engineering of hybrid microwires enables a sustained release of the anti-inflammatory agent over extended periods of time, thus paving the way to fabricate neuroprosthetic devices capable of attenuating the foreign body response.

Giant Island Mice Exhibit Widespread Gene Expression Changes in Key Metabolic Organs.

Island populations repeatedly evolve extreme body sizes, but the genomic basis of this pattern remains largely unknown. To understand how organisms on islands evolve gigantism, we compared genome-wide patterns of gene expression in Gough Island mice, the largest wild house mice in the world, and mainland mice from the WSB/EiJ wild-derived inbred strain. We used RNA-seq to quantify differential gene expression in three key metabolic organs: gonadal adipose depot, hypothalamus, and liver. Between 4,000 and 8,800 genes were significantly differentially expressed across the evaluated organs, representing between 20% and 50% of detected transcripts, with 20% or more of differentially expressed transcripts in each organ exhibiting expression fold changes of at least 2×. A minimum of 73 candidate genes for extreme size evolution, including Irs1 and Lrp1, were identified by considering differential expression jointly with other data sets: 1) genomic positions of published quantitative trait loci for body weight and growth rate, 2) whole-genome sequencing of 16 wild-caught Gough Island mice that revealed fixed single-nucleotide differences between the strains, and 3) publicly available tissue-specific regulatory elements. Additionally, patterns of differential expression across three time points in the liver revealed that Arid5b potentially regulates hundreds of genes. Functional enrichment analyses pointed to cell cycling, mitochondrial function, signaling pathways, inflammatory response, and nutrient metabolism as potential causes of weight accumulation in Gough Island mice. Collectively, our results indicate that extensive gene regulatory evolution in metabolic organs accompanied the rapid evolution of gigantism during the short time house mice have inhabited Gough Island.

The Number of Transcription Factors at an Enhancer Determines Switch-like Gene Expression

NF-κB is a transcription factor that activates super enhancers (SEs) and typical enhancers (TEs) and triggers threshold and graded gene expression, respectively. However, the mechanisms by which NF-κB selectively participates in these enhancers remain unclear. Here we show using mouse primary B lymphocytes that SE activity simultaneously associates with chromatin opening and enriched NF-κB binding, resulting in a higher fold change and threshold expression upon B cell receptor (BCR) activation. The higher fold change results from longer DNA, whereas the threshold response is explained by synergy in DNA-NF-κB binding and is supported by the coexistence of PU.1 and NF-κB in a SE before cell stimulation. This model indicates that the pre-existing NF-κB functions as a seed and triggers its processive binding upon BCR activation. Our mathematical modeling of the single-cell transcriptome reveals an additional role for SEs in divergent clonal responses in B cells.

Biological correlates to support a clinical role for tagraxofusp, a novel targeted therapy directed to CD123, in combination with pomalidomide and dexamethasone, to target plasmacytoid dendritic cells in poor-risk patients with multiple myeloma.

e20537 Background: We have previously described the pivotal pathogenetic role of plasmacytoid dendritic cells (pDCs; CD123/IL-3Ra+) in multiple myeloma (MM) (Chauhan et al Cancer Cell, 2009,16:309). In preclinical MM models, we demonstrated that tagraxofusp, a novel targeted therapy directed to CD123, triggers anti-MM activity by reducing the viability of MM-promoting pDCs. These observations led to an ongoing phase 1/2 clinical trial of tagraxofusp in MM patients (NCT02661022). The treatment regimen demonstrated safety and efficacy, with 5 of 9 heavily pretreated patients achieving durable partial response (PR). Here, we report the initial results of our translational exploratory studies using bone marrow (BM), peripheral blood (PB), and serum from the study cohort. Methods: pDCs and MM tumor cells were purified from BM/PB samples and quantified using FACS, as described ( Ray et al Leukemia, 2018,32:843). A high-throughput seroproteomics platform SOMAscan was utilized to analyze 1,310 protein analytes in serum samples from MM patients (N = 9). SOMAscan data were subjected to meta-analysis to generate heatmaps, followed by hierarchical cluster analysis. SOMAscan results were validated with ELISA using supernatants from MM patient pDCs cultured with or without tagraxofusp. Results: Analysis of BM/PB samples from MM patients receiving tagraxofusp therapy showed a marked reduction in the frequency of viable pDCs [average 2% at screen vs 0.75% post-tagraxofusp; N = 6; p = 0.036]. pDCs isolated from tagraxofusp-treated patients showed decreased ability to trigger MM cell growth. Seroproteomics analysis of MM patient serum before and after tagraxofusp therapy showed alterations in the levels of 100 proteins [Median Fold Change in expression: 0.39 to 4.5; n = 6; 3 each; p &lt; 0.05]. Importantly, we found that tagraxofusp treatment reduced pDC-related soluble proteins, in particular, IFN-a and IL-3Rα. Additionally, our earlier study showed that pDC-MM interactions triggered secretion of MM cell growth including IL-3, which serves a dual role of promoting pDC survival and MM cell growth. Importantly, tagraxofusp decreased serum IL-3, suggesting that tagraxofusp attenuates survival mechanisms for tumor-promoting pDCs. Conclusions: Our current correlative studies validate target specificity of tagraxofusp and support further evaluation for this novel therapeutic to improve the clinical outcome of patients with MM.

Profiling of the Peripheral Blood Mononuclear Cell Proteome in Schizophrenia and Mood Disorders for the Discovery of Discriminatory Biomarkers: A Proof-of-Concept Study

Introduction: Current diagnoses in psychiatry are solely based on the evaluation of clinical presentation by the treating psychiatrist. This results in a high percentage of misdiagnosis and consequential inefficient treatment; especially regarding major depressive disorder (MDD), depression in the context of bipolar disorder (BD-D), bipolar disorder with manic symptoms (BD-M), and psychosis in the context of schizophrenia (SZ). Objective biomarkers allowing for accurate discriminatory diagnostics are therefore urgently needed. Methods: Peripheral blood mononuclear cell (PBMC) proteomes of patients with MDD (n = 5) , BD-D (n = 3), BD-M (n = 4), and SZ (n = 4), and also of healthy controls (HC; n = 6) were analyzed by state-of-the-art mass spectrometry. Proteins with a differential expression of a >2 standard deviation (SD) expression fold change from that of the HC and between either MDD versus BD-D or BD-M versus SZ were subsequently identified as potential discriminatory biomarkers. Results: In total, 4,271 individual proteins were retrieved from the HC. Of these, about 2,800 were detected in all patient and HC samples. For objective discrimination between MDD and BD-D, 66 candidate biomarkers were found. In parallel, 72 proteins might harbor a biomarker capacity for differential diagnostics of BD-M and SZ. A single biomarker was contraregulated versus HC in each pair of comparisons. Discussion: With this work, we provide a register of candidate biomarkers with the potential to objectively discriminate MDD from BD-D, and BD-M from SZ. Although concerning a proof-of-concept study with limited sample size, these data provide a stepping-stone for follow-up research on the validation of the true discriminatory potential and feasibility of clinical implementation of the discovered biomarker candidates.

Neuroendocrine Tumor Omic Gene Cluster Analysis Amplifies the Prognostic Accuracy of the NETest

Background: The NETest is a multigene assay comprising 51 circulating neuroendocrine tumor (NET)-specific transcripts. The quotient of the 51-gene assay is based upon an ensemble of machine learning algorithms. Eight cancer hallmarks or “omes” (apoptome, epigenome, growth factor signalome, metabolome, proliferome, plurome, secretome, SSTRome) represent 29 genes. The NETest is an accurate diagnostic (>90%) test, but its prognostic utility has not been assessed. In this study, we describe the expansion of the NETest omic cluster components and demonstrate that integration amplifies NETest prognostic accuracy. Methods: Group 1: n = 222; including stable disease (SD, n = 146), progressive disease (PD, n = 76), and controls (n = 139). Group 2: NET Registry NCT02270567; n = 88; prospective samples (SD, n = 54; PD, n = 34) with up to 24 months follow-up. We used PubMed literature review, interactomic analysis, nonparametric testing, Kaplan-Meier survival curves, and χ² analyses to inform and define the prognostic significance of NET genomic “hallmarks.” Results: 2020 analyses: In-depth analyses of 47 ­NETest genes identified a further six omes: fibrosome, inflammasome, metastasome, NEDome, neurome, and TFome. Group 1 analysis: Twelve omes, excluding the inflammasome and apoptome, were significantly (p < 0.05, 2.1- to 8.2-fold) elevated compared to controls. In the PD group, seven omes (proliferome, NEDome, epigenome, SSTRome, neurome, metastasome, and fibrosome) were elevated (both expression levels and fold change >2) versus SD. Group 2 analysis: All these seven omes were upregulated. In PD, they were significantly more elevated (p < 0.02) than in SD. The septet omic expression exhibited a 69% prognostic accuracy. The NETest alone was 70.5% accurate. A low NETest (≤40) integrated with epigenome/metastasome levels was an accurate prognostic for PD (90%). A high NETest (>40) including the fibrosome/NEDome predicted PD development within 3 months (100%). Using decision tree analysis to integrate the four omes (epigenome, metastasome, fibrosome, and NEDome) with the NETest score generated an overall prognostic accuracy of 93%. Conclusions: Examination of NETest omic gene cluster analysis identified five additional clinically relevant cancer hallmarks. Identification of seven omic clusters (septet) provides a molecular pathological signature of disease progression. The integration of the quartet (epigenome, fibrosome, metastasome, NEDome) and the NETest score yielded a 93% accuracy in the prediction of future disease status.

Bacterially-derived indole modulates murine arthritis through alteration of B cell glycosylation enzymes

Abstract Indole, a bacteria-derived immunomodulatory metabolite that binds the aryl hydrocarbon receptor (AhR), is produced by bacterial tryptophanase in the intestinal tract of both mice and humans. Our studies using the murine collagen-induced arthritis (CIA) model of inflammatory arthritis demonstrated that intestinal bacteria influence disease severity through modulation of autoantibody glycosylation, and indole significantly correlated with the development of disease (P&amp;lt;0.0001). Therefore, we hypothesized that indole modulates CIA through modulation of B cell glycosylation enzymes via AhR signaling. Mice with CIA that were protected by treatment with broad-spectrum antibiotics at the time of booster injection (CIA+Abx), lost the protective effect of antibiotics when dosed with indole (arthritis score 3.6±0.75 CIA+Abx+Indole vs. 1±0.00 CIA+Abx, p&amp;lt;0.001). Mice with CIA, compared to CIA+Abx, demonstrated significantly increased expression of β1,4-galactosyltransferase 1 (GalT) and β-galactoside α2, 6-sialyltransferase 1 (St6gal1), two enzymes important in mediating pathogenic antibody glycosylation; this effect was reversed in CIA+Abx+Indole. We further demonstrate that Ramos cells, a human B cell line, upregulate glycosylation enzymes and AhR expression in response to indole exposure, with a fold change expression of 3.315±0.92 of B4GALT5 in indole-treated cells as compared to untreated (p=0.05). While the requirement of AhR and the glycosylation status of antibodies in our studies need to be confirmed, these results suggest that microbiome changes in CIA result in indole signaling on B cells, affecting glycosylation enzymes that modulate autoantibody effector function towards a proinflammatory profile.

Mitochondrial Permeability Transition Induces Atrophy in C2C12 Myotubes

INTRODUCTIONIn advanced age (&gt;75 yrs), muscle atrophy can lead to physical frailty, impaired mobility, and increased mortality. Impairment and functional decline of mitochondria within skeletal muscle are seen with aging, including a sensitization to mitochondrial permeability transition (MPT). MPT is an established pathological mechanism causing ischemic organ damage and has been observed in aged motoneurons and skeletal muscle. However, the role of MPT in age‐related muscle atrophy has not been determined. To better understand how MPT may induce skeletal muscle atrophy, we determined the impact of inducing MPT on myotube size in a cell culture model and the impact of modulating MPT on classical atrophy signaling transcripts. We hypothesized that induction of MPT in myotubes would cause atrophy, and MPT would modulate atrophy‐related signaling pathways.METHODSTo determine if MPT can induce myotube atrophy, C2C12 myotubes were treated with doxorubicin (Dox; 1 μM), a known inducer of MPT, or DMSO (vehicle, 1 μM) on day 4 post‐differentiation for 12, 24, and 48 h. At each time point, cells were fixed/permeabilized in icecold methanol/acetone (1:1), blocked, and incubated with a primary antibody against sarcomeric myosin (MF‐20). Cells were incubated with a fluorescent secondary antibody and imaged at 20X on an inverted fluorescent microscope. Images (n=4 per well, 5 wells/treatment) were analyzed to quantify MF20+ area (myotube area) using a custom written program in CellProfiler.To assess signaling mechanisms downstream of MPT, C2C12 myotubes were treated with Dox (1μM), Dox + an inhibitor of MPT (bongkrekic acid [BA]; DOX+BA, each 1 μM), or DMSO (vehicle;1 μM) for 12 h on day 4 post‐differentiation (n=3 biological replicates; n=4 technical replicates per treatment). At 12 h post‐treatment, cells were collected in TRIzol and their RNA isolated for RT‐PCR analysis of transcriptional regulators of muscle atrophy (FOXO1, NfKB1, Gadd45a, ATF4), an autophagy‐regulating gene (p62/SQSTM), and E3 ubiquitin ligases (Mafbx, MuRF). The comparative threshold cycle (CT) method was used to calculate fold changes in expression with 18s as an endogenous control. Relative fold changes in expression are presented as 2−ΔΔCT and normalized to DMSO‐treated samples.Two‐tailed Student’s t‐tests were used to assess differences between DOX vs. DMSO, and DOX vs. DOX+BA, respectively. Significance was accepted at P&lt;0.05.RESULTSDOX treatment significantly reduced myotube area by 24 h post‐treatment, and this persisted through 48 h (P&lt;0.05). Inhibiting MPT with BA attenuated mRNA levels of transcription factors FOXO1 and ATF4, Gadd45a, the atrogene MuRF1, and the autophagy regulating gene p62 at 12 h post‐treatment (P&lt;0.05 for all). There was no difference between DOX vs. DOX+BA for MAFbx (P=0.076) or NfKB1 (0.692) expression.DISCUSSIONConsistent with our hypothesis, induction of MPT with DOX caused myotube atrophy. Additionally, inhibiting MPT in the presence of DOX attenuated mRNA levels of classical atrophy signaling pathway components, including FOXO1, ATF4, Gadd45α, MuRF1 and p62. Thus, our results suggest that MPT is a cause of atrophy in aging skeletal muscle.Support or Funding InformationSB is supported by a T32 from the National Institute of Child Health and Human Development Grant T32HD043730.

Hypoxia Susceptibility Related to the Expression of Long Noncoding RNA as Regulators of Abraxas2 and Camk2d in the Heart Tissue of a Hypoxic Mouse Model

Long noncoding RNAs (lncRNAs) play a regulatory role in hypoxic disease states, and identifying regulators that control gene expression in a hypoxic mouse model will introduce therapeutic targets and biomarkers of susceptibility to hypoxia. Previous studies show that CD‐1 and C57BL/6J mice are sensitive and resistant to hypoxic stress, respectively.CD‐1 and C57BL/6J mice were subjected to an intraperitoneal injection of SE‐175 (0.7 mg/kg) to induce a hypoxic response or 1x phosphate‐buffered saline (PBS) and then sacrificed after five hours in normoxic condition. RNA was extracted from heart tissue and analyzed in a microarray. Genomic BLAST was performed between the human genome and mice lncRNAs with ±2 fold change in expression (p &lt; 0.05). The expression of several genes coding sequences and their associated lncRNAs were analyzed by qPCR.We identified two lncRNA 1500002F19Rik and LOC108168899 that were found in close proximity to the genes Abraxas2 and Camk2d, respectively. Microarray results suggested that 1500002F19Rik and LOC108168899 may play a role in the expression of Abraxas2 and Camk2d, respectively. The expression levels of Abraxas2, Camk2d, and their corresponding lncRNA were analyzed using qPCR. In mice treated with SE‐175, Abraxas2 and 1500002F19Rik were both upregulated in C57BL/6J mice, but neither displayed a significant change in CD‐1 mice. Camk2d and LOC108168899 were both upregulated in C57BL/6J mice, while Camk2d was upregulated in CD‐1 mice, but LOC108168899 showed no significant change.Because of the cytoprotective effects of Abraxas2 against oxidative stress, its upregulation and that of 1500002F19Rik in C57BL/6J mice provides a possible mechanism for their resistance to hypoxic stress. Camk2d is implicated in oxidative stress in cardiomyocytes and was upregulated to a higher magnitude in CD‐1 mice than in C57BL/6J mice, while LOC108168899 only showed increased expression in C57BL/6J mice. This suggests that LOC108168899 can be involved in downregulation of Camk2d that prevents further oxidative stress in C57BL/6J mice, so its lower expression in CD‐1 mice can lead to higher expression of Camk2d which may contribute to susceptibility to hypoxia.Support or Funding InformationThis project was supported by Touro University Nevada research grant.

Differentially expressed mRNAs and lncRNAs shared between activated human hepatic stellate cells and nash fibrosis

We previously reported dysregulated expression of liver-derived messenger RNA (mRNA) and long noncoding RNA (lncRNA) in patients with advanced fibrosis resulting from nonalcoholic fatty liver disease (NAFLD). Here we sought to identify changes in mRNA and lncRNA levels associated with activation of hepatic stellate cells (HSCs), the predominant source of extracellular matrix production in the liver and key to NAFLD-related fibrogenesis. We performed expression profiling of mRNA and lncRNA from LX-2 cells, an immortalized human HSC cell line, treated to induce phenotypes resembling quiescent and myofibroblastic states. We identified 1964 mRNAs (1377 upregulated and 587 downregulated) and 1460 lncRNAs (665 upregulated and 795 downregulated) showing statistically significant evidence (FDR ≤0.05) for differential expression (fold change ≥|2|) between quiescent and activated states. Pathway analysis of differentially expressed genes showed enrichment for hepatic fibrosis (FDR = 1.35E-16), osteoarthritis (FDR = 1.47E-14), and axonal guidance signaling (FDR = 1.09E-09). We observed 127 lncRNAs/nearby mRNA pairs showing differential expression, the majority of which were dysregulated in the same direction. A comparison of differentially expressed transcripts in LX-2 cells with RNA-sequencing results from NAFLD patients with or without liver fibrosis revealed 1047 mRNAs and 91 lncRNAs shared between the two datasets, suggesting that some of the expression changes occurring during HSC activation can be observed in biopsied human tissue. These results identify lncRNA and mRNA expression patterns associated with activated human HSCs that appear to recapitulate human NAFLD fibrosis.