TaqMan Low Density Array Research Articles

Eicosatetraynoic Acid Regulates Pro-Fibrotic Pathways in an Induced Pluripotent Stem Cell Derived Macrophage:Human Intestinal Organoid Model of Crohn's Disease.

We previously identified small molecules predicted to reverse an ileal gene signature for future Crohn's Disease (CD) strictures. Here we used a new human intestinal organoid (HIO) model system containing macrophages to test a lead candidate, eicosatetraynoic acid (ETYA). Induced pluripotent stem cell lines (iPSC) were derived from CD patients and differentiated into macrophages and HIOs. Macrophages and macrophage:HIO co-cultures were exposed to lipopolysaccharide (LPS) with and without ETYA pre-treatment. Cytospin and flow cytometry characterized macrophage morphology and activation markers, and RNA sequencing defined the global pattern of macrophage gene expression. TaqMan Low Density Array, Luminex multiplex assay, immunohistologic staining, and sirius red polarized light microscopy were performed to measure macrophage cytokine production and HIO pro-fibrotic gene expression and collagen content. iPSC-derived macrophages exhibited morphology similar to primary macrophages and expressed inflammatory macrophage cell surface markers including CD64 and CD68. LPS-stimulated macrophages expressed a global pattern of gene expression enriched in CD ileal inflammatory macrophages and matrisome secreted products, and produced cytokines and chemokines including CCL2, IL1B, and OSM implicated in refractory disease. ETYA suppressed CD64 abundance and pro-fibrotic gene expression pathways in LPS stimulated macrophages. Co-culture of LPS-primed macrophages with HIO led to up-regulation of fibroblast activation genes including ACTA2 and COL1A1, and an increase in HIO collagen content. ETYA pre-treatment prevented pro-fibrotic effects of LPS-primed macrophages. ETYA inhibits pro-fibrotic effects of LPS-primed macrophages upon co-cultured HIO. This model may be used in future untargeted screens for small molecules to treat refractory CD.

Evaluation of circulating microRNA signature in patients with erosive hand osteoarthritis: The HOAmiR study

ObjectivesTo identify circulating micro-RNAs differentially expressed in patients with erosive hand osteoarthritis (HOA) compared to patients with non-erosive HOA and patients without HOA. MethodsIn the screening phase, 768 well-characterized micro-RNAs using Taqman Low Density Array cards were measured in 30 sera from 10 patients with erosive HOA, 10 patients with non-erosive HOA and 10 controls without HOA, matched for age and BMI. In a second step, we validated the micro-RNAs identified at the screening phase (adjusted p value < 0.05 after false discovery rate correction using Benjamini-Hochberg method and literature review) in larger samples (60 patients with erosive HOA and 60 patients without HOA matched for age and BMI). ResultsIn the screening phase, we identified 21 down-regulated and 4 up-regulated micro-RNAs of interest between erosive HOA and control groups. Among these, 9 micro-RNAs (miR-373-3p, miR-558, miR-607, miR-653-5p, miR-137 and miR448 were down-regulated and miR-142-3p, miR-144-3p and miR-34a-5p were up-regulated) were previously described in chondrocytes homeostasis or OA. We found only one significantly down-regulated micro-RNA between erosive and non-erosive HOA. In the validation phase, we showed replication of a single micro-RNA the significant downregulation of miR-196-5p, that had been previously identified in the screening phase among patients with erosive HOA compared to those without HOA.After reviewing the literature and the miRNA-gene interaction prediction model, we found that this microRNA could interact with bone homeostasis and HOXC8, which could explain its role in osteoarthritis. ConclusionsWe found that miR-196-5p was down regulated in patients with erosive HOA and some of its targets could explain a role in OA.

Regulatory and memory T lymphocytes infiltrating prostate tumors predict long term clinical outcomes.

The localization, density but mostly the phenotype of tumor infiltrating lymphocytes (TIL) provide important information on the initial interaction between the host immune system and the tumor. Our objective was to assess the prognostic significance of T (CD3+), T regulatory (Treg) (FoxP3+) and T memory (Tmem) (CD45RO+) infiltrating lymphocytes and of genes associated with TIL in prostate cancer (PCa). Immunohistochemistry (IHC) was used to assess the infiltration of CD3+, FoxP3+ and CD45RO+ cells in the tumor area, tumor margin and adjacent normal-like epithelium of a series of 98 PCa samples with long clinical follow-up. Expression of a panel of 31 TIL-associated genes was analyzed by Taqman Low-Density Array (TLDA) technology in another series of 50 tumors with long clinical follow-up. Kaplan-Meier and Cox proportional hazards regression analyses were performed to determine association of these markers with biochemical recurrence (BCR), need for definitive androgen deprivation therapy (ADT) or lethal PCa. TIL subtypes were present at different densities in the tumor, tumor margin and adjacent normal-like epithelium, but their density and phenotype in the tumor area were the most predictive of clinical outcomes. In multivariate analyses, a high density of Treg (high FoxP3+/CD3+ cell ratio) predicted a higher risk for need of definitive ADT (HR=7.69, p=0.001) and lethal PCa (HR=4.37, p=0.04). Conversely, a high density of Tmem (high CD45RO+/CD3+ cell ratio) predicted a reduced risk of lethal PCa (HR=0.06, p=0.04). TLDA analyses showed that a high expression of FoxP3 was associated with a higher risk of lethal PCa (HR=5.26, p=0.02). Expression of CTLA-4, PD-1, TIM-3 and LAG-3 were correlated with that of FoxP3. Amongst these, only a high expression of TIM-3 was associated with a significant higher risk for definitive ADT in univariate Cox regression analysis (HR=3.11, p=0.01). These results show that the proportion of Treg and Tmem found within the tumor area is a strong and independent predictor of late systemic progression of PCa. Our results also suggest that inhibition of TIM-3 might be a potential approach to counter the immunosuppressive functions of Treg in order to improve the anti-tumor immune response against PCa.

MicroRNA-138 inhibits hypoxia-inducible factor 1α expression in breast cancer cells

Background Hypoxia, a critical feature during cancer development, leads to the stabilization and activation of the hypoxia-inducible factor 1-alpha (HIF-1α) to drive the expression of many target genes which in turn can promote many aspects of breast cancer biology, mainly metastasis and resistance to therapy. MicroRNAs are known to modulate the expression of many genes involved in breast cancer tumorigenesis. In this study, we examined the regulatory effect of miRNAs on HIF1α expression. Methods MCF-7 and MDA-MB-231 were cultivated under normoxia or hypoxia conditions. TaqMan-Low Density Array (TLDA) was used to characterize the miRNA signatures. Wild-Type (WT) or mutated fragments of HIF-1α 3’UTR containing the miR-138 potential target site were cloned downstream of the Renilla luciferase gene in the psiCHECK-1 plasmid. Luciferase assays were then carried out. A lentiviral vector containing copGFP as a reporter gene was prepared and transduced into MCF-7 and MDA-MB-231 cells to assess the effect of identified deregulated miRNAs on HIF-1α expression. Results Under hypoxic conditions, MCF-7 cells showed deregulated expression for 12 miRNAs. In the case of MDA-MB-231 cells, 16 miRNAs were deregulated in response to hypoxia. Interestingly, miR-138 that was downregulated in both MCF-7 and MDA-MB-231 cells cultivated under hypoxic conditions appeared to have a binding site in 3’UTR of HIF-1α. Moreover, our results indicated that miR-138 could down regulate HIF-1α expression, upon binding directly to its 3’UTR. Conclusions Interestingly, our data highlights miR-138 as a potential therapeutic target to reduce HIF-1α expression and subsequently restrain breast cancer invasion and metastasis.

Differentiation and immunosuppressive function of CD19+CD24hiCD27+ regulatory B cells are regulated through the miR-29a-3p/NFAT5 pathway

BackgroundRegulatory B cells (Bregs) is an indispensable element in inducing immune tolerance after liver transplantation. As one of the microRNAs (miRNAs), miR-29a-3p also inhibits translation by degrading the target mRNA, and yet the relationship between Bregs and miR-29a-3p has not yet been fully explored. This study aimed to investigate the impact of miR-29a-3p on the regulation of differentiation and immunosuppressive functions of memory Bregs (mBregs) and ultimately provide potentially effective therapies in inducing immune tolerance after liver transplantation. MethodsFlow cytometry was employed to determine the levels of Bregs in peripheral blood mononuclear cells. TaqMan low-density array miRNA assays were used to identify the expression of different miRNAs, electroporation transfection was used to induce miR-29a-3p overexpression and knockdown, and dual luciferase reporter assay was used to verify the target gene of miR-29a-3p. ResultsIn patients experiencing acute rejection after liver transplantation, the proportions and immunosuppressive function of mBregs in the circulating blood were significantly impaired. miR-29a-3p was found to be a regulator of mBregs differentiation. Inhibition of miR-29a-3p, which targeted nuclear factor of activated T cells 5 (NFAT5), resulted in a conspicuous boost in the differentiation and immunosuppressive function of mBregs. The inhibition of miR-29a-3p in CD19+ B cells was capable of raising the expression levels of NFAT5, thereby promoting B cells to differentiate into mBregs. In addition, the observed enhancement of differentiation and immunosuppressive function of mBregs upon miR-29a-3p inhibition was abolished by the knockdown of NFAT5 in B cells. ConclusionsmiR-29a-3p was found to be a crucial regulator for mBregs differentiation and immunosuppressive function. Silencing miR-29a-3p could be a potentially effective therapeutic strategy for inducing immune tolerance after liver transplantation.

Transcriptomic Analysis of Alzheimer's Disease Pathways in a Pakistani Population.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder that is most prevalent in elderly individuals, especially in developed countries, and its prevalence is now increasing in developing countries like Pakistan. Our goal was to characterize key genes and their levels of expression and related molecular transcriptome networks associated with AD pathogenesis in a pilot case-control study in a Pakistani population. To obtain the spectrum of molecular networks associated with pathogenesis in AD patients in Pakistan (comparing cases and controls), we used high-throughput qRT-PCR (TaqMan Low-Density Array; n = 33 subjects) coupled with Affymetrix Arrays (n = 8) and Ingenuity Pathway Analysis (IPA) to identify signature genes associated with Amyloid processing and disease pathways. We confirmed 16 differentially expressed AD-related genes, including maximum fold changes observed in CAPNS2 and CAPN1. The global gene expression study observed that 61% and 39% of genes were significantly (p-value 0.05) up- and downregulated, respectively, in AD patients compared to healthy controls. The key pathways include, e.g., Amyloid Processing, Neuroinflammation Signaling, and ErbB4 Signaling. The top-scoring networks in Diseases and Disorders Development were Neurological Disease, Organismal Injury and Abnormalities, and Psychological Disorders. Our pilot study offers a non-invasive and efficient way of investigating gene expression patterns by combining TLDA and global gene expression method in AD patients by utilizing whole blood. This provides valuable insights into the expression status of genes related to Amyloid Processing, which could play potential role in future studies to identify sensitive, early biomarkers of AD in general.

Decreased plasma miR-140-3p is associated with coronary artery disease

BackgroundAlthough many circulating miRNAs (c-miRNAs) are associated with coronary artery disease (CAD), they are far from being the biomarker for CAD diagnosis or risk prediction. Therefore, novel c-miRNAs discovery and validation are still required, especially evaluating their prediction capacity. ObjectivesIdentify novel CAD-related c-miRNAs and evaluate its risk prediction capacity for CAD. Methods: miRNAs associated with CAD were preliminarily investigated in three paired samples representing pre-CAD stage and CAD stage of three female individuals using the Applied Biosystems miRNA TaqMan® Low-Density Array (TLDA). Then, the candidate miRNAs were further verified in an independent case-control study including 129 CAD patients and 76 controls, and their potential practical value in prediction for CAD was evaluated using a machine learning (ML) algorithm. The accuracy of classification and prediction was assessed with the area under the receiver operating characteristic curve (AUC). ResultsTLDA analysis shows that miR-140-3p decreased significantly in CAD-stage (FC = −3.01, P = 0.007). Further study shows that miR-140-3p was significantly lower in CAD group [1.26 (0.68, 2.01)] than in control group [2.07 (1.19, 3.21)] (P < 0.001) and independently associated with CAD (P < 0.001). The addition of miR-140-3p to the variables including smoking history, HDL-c, and APOA1 improved the accuracy of classification by logistic regression and of prediction for CAD by ML models. The ML models built with miR-140-3p and HDL-c, respectively, had a similar prediction accuracy. The feature importance of miR-140-3p and HDL-c in the ML models was also similar. Decision curve analysis showed that miR-140-3p and HDL-c had almost identical net benefits. ConclusionReduced levels of miR-140-3p is linked to CAD, and it is possible to use the plasma level of miR-140-3p as a means of evaluating the risk of CAD.

Profiling of microRNA as a tool to introduce rAAV vectors in gene therapy of breast cancer: A preliminary report.

Despite the wide range of diagnostic and therapeutic methods, breast cancer is responsible for many deaths each year. One of the original and novel cancer therapeutic approaches is gene therapy based on recombinant adeno-associated viral vectors. Among the molecular factors with the potential to become useful diagnostic biomarkers, microRNA (miRNA) molecules are being considered for personalized therapies. The aim of the study was to examine the utility of miRNA profiling in the design of personalized recombinant adeno-associated virus (rAAV)-based gene therapy for breast cancer patients. The analysis of 754 miRNAs in 7 breast cancer samples and control samples was performed using real-time polymerase chain reaction (PCR) based on TaqMan® Low-density Array (TLDA) cards. Online repositories were used to explore the relationship between miRNAs and genes encoding rAAV receptors (KIAA0319L, HSPG2, FGFR1, c-MET, PDGFRA, ITGB5, and RPSA). Then, we performed a comparative analysis of the results to examine the possibility of using miRNA profiling in the design of rAAV-based therapeutic protocols. Fifty-two percent of tested miRNAs were noted in at least 1 analyzed breast cancer and control tissue. Thirteen miRNAs were selected due to being outliers in the tested samples. In total, 155 miRNAs targeted genes encoding rAAV receptors in the tested samples (29 miRNAs for KIAA0319L, 60 miRNAs for c-MET, 31 miRNAs for HSPG2, 43 miRNAs for FGFR1, 36 miRNAs for PDGFRA, 18 miRNAs for RPSA, and 25 miRNAs for ITGB5). The expression of the selected miRNAs was not homogeneous across the 7 samples. Profiling of microRNA could be a significant factor in the design of rAAV-based personalized gene therapy for breast cancer patients.

High infiltration of CD209+ dendritic cells and CD163+ macrophages in the peritumor area of prostate cancer is predictive of late adverse outcomes.

Prostate cancer (PCa) shows considerable variation in clinical outcomes between individuals with similar diseases. The initial host-tumor interaction as assessed by detailed analysis of tumor infiltrating immune cells within the primary tumor may dictate tumor evolution and late clinical outcomes. In this study, we assessed the association between clinical outcomes and dendritic cell (DC) or macrophage (MΦ) tumor infiltration as well as with expression of genes related to their functions. Infiltration and localization of immature DC, mature DC, total MΦ and M2-type MΦ was analyzed by immunohistochemistry in 99 radical prostatectomy specimens from patients with 15.5 years median clinical follow-up using antibodies against CD209, CD83, CD68 and CD163, respectively. The density of positive cells for each marker in various tumor areas was determined. In addition, expression of immune genes associated with DC and MΦ was tested in a series of 50 radical prostatectomy specimens by Taqman Low-Density Array with similarly long follow-up. Gene expression was classified as low and high after unsupervised hierarchical clustering. Numbers and ratio of positive cells and levels of gene expression were correlated with endpoints such as biochemical recurrence (BCR), need for definitive androgen deprivation therapy (ADT) or lethal PCa using Cox regression analyses and/or Kaplan-Meier curves. Positive immune cells were observed in tumor, tumor margin, and normal-like adjacent epithelium areas. CD209+ and CD163+ cells were more abundant at the tumor margin. Higher CD209+/CD83+ cell density ratio at the tumor margin was associated with higher risk of ADT and lethal PCa while higher density of CD163+ cells in the normal-like adjacent epithelium was associated with a higher risk of lethal PCa. A combination of 5 genes expressed at high levels correlated with a shorter survival without ADT and lethal PCa. Among these five genes, expression of IL12A and CD163 was correlated to each other and was associated with shorter survival without BCR and ADT/lethal PCa, respectively. A higher level of infiltration of CD209+ immature DC and CD163+ M2-type MΦ in the peritumor area was associated with late adverse clinical outcomes.

O-062 MicroRNAs in Blastocoel Fluid: a molecular signature for predicting human embryo implantation potential

Abstract Study question Can microRNA expression in blastocoel fluid (BF) predict embryo implantation potential? Summary answer Up-regulation of miR-106, miR-373, miR-301, miR-320 and miR-525-3p represents the molecular signature characterizing the embryos with high implantation capability. What is known already The discovery of DNA within the BF and in spent embryo culture media, has caused increased interest in the non-invasive preimplantation testing application for monogenic disorders and aneuploidies. Aneuploid embryos have a lower implantation potential, however, chromosomal abnormalities represent only a tiny percentage of the causes of implantation failure. MiRNAs can control all cellular pathways and are involved in pluripotency, self-renewal, and stemness, and their altered regulation affects different human diseases. Study design, size, duration From September 2018 to March 2022, 112 BFsampleswere collected from human embryos on the fifth day of development, before the blastocyst cryopreservation. The samples were classified according to blastocyst grade and the data on implantation outcome and term births were collected for the transferred embryos. We compared the expression profiles of 89 miRNAs, previously identified in BF, between 33 BF from implanted embryos and 30 from non-implanted ones, regardless of blastocyst grade. Participants/materials, setting, methods By custom-designed TaqMan Low-Density Array card (TLDA), we analyzed the expression of 89 miRNAs in 4 different BF samples simultaneously. Differentially expressed (DE) miRNAs were identified by Volcano plot and Significance Analysis of Microarrays (SAM) tests. Bioinformatic analysis was performed to identify the biological role of the DE miRNAs. To evaluate miRNA’s ability for predicting implantation, Pearson’s correlation analyses, classical univariate Receiver Operator Characteristic (ROC) curve analysis, and the optimal cut-off value determination were performed. Main results and the role of chance We found five miRNAs, miR-106, miR-373, miR-301, miR-320 and miR-525-3pup-regulated in BF from implanted blastocysts. The identified miRNAs perform an important role during the first phases of embryo development suggesting that their up-regulation may reflect embryo health. Moreover, four of the five miRNAs showed significant correlation coefficients in both implanted and non-implanted blastocysts, indicating that their expression changes in the same way in the single sample and reflects the potentiality of the embryo to implant. Finally, ROC curve analysis confirmed that our miRNAs could be considered potential biomarkers for implantation. Limitations, reasons for caution Successful implantation requires a close dialogue between the embryo and the endometrium, mediated by different proteins and miRNAs produced by both the embryo and maternal tissues. Determining the quality of the embryo and its implantation potential is not sufficient to predict successful pregnancy outcomes. Wider implications of the findings This study represents the first report correlating miRNA profiles in BFand implantation and suggests that miRNA signature could become an accurate tool to evaluate embryo quality. It could be associated or replaced with the PGT-A to choose the most competent embryo and improve the outcome of assisted reproduction cycles. Trial registration number The study has been approved by theEthical Committee of Azienda Ospedaliero Universitaria Policlinico &amp;quot;G.Rodolico -San Marco&amp;quot; Catania.

Medium- and time-related effects on hypothermic storage of rat testicular cells.

Testicular samples obtained for fertility preservation often need to be transported between clinics. This study aimed to mimic this short-term hypothermic storage (4-8 °C) and explore the impact of these conditions and the transport medium composition on prepubertal rat testicular tissue samples. Testicular tissue samples obtained from seven days post-partum rats were transferred to six compositionally different basal culture media and a balanced salt solution, which had been kept at 4-8 °C prior to transfer. The samples were preserved for either 12 or 24 hours in these hypothermic conditions. The potential effects of the short-term storage were evaluated by assessing the morphology, measuring the testosterone levels by radioimmunoassay and analysing 96 genes with TaqMan Low-Density Arrays. Summarizing results: Levels of gene expression related to energy, apoptosis and angiogenesis pathways were altered after hypothermic storage for 12 and especially 24 hours. We observed only minor differences in gene expression profiles for germ and testicular somatic cells, and no differences in tissue morphology and testosterone production levels. Short-term hypothermic storage of testicular tissue with a maximum duration of 24 hours does not affect the overall expression profile of testicular cell-specific genes; however, in a minor way, it affects the expression of specific cellular genes.

Technical development and validation of a clinically applicable microenvironment classifier as a biomarker of tumour hypoxia for soft tissue sarcoma

BackgroundSoft tissue sarcomas (STS) are rare, heterogeneous tumours and biomarkers are needed to inform management. We previously derived a prognostic tumour microenvironment classifier (24-gene hypoxia signature). Here, we developed/validated an assay for clinical application.MethodsTechnical performance of targeted assays (Taqman low-density array, nanoString) was compared in 28 prospectively collected formalin-fixed, paraffin-embedded (FFPE) biopsies. The nanoString assay was biologically validated by comparing to HIF-1α/CAIX immunohistochemistry (IHC) in clinical samples. The Manchester (n = 165) and VORTEX Phase III trial (n = 203) cohorts were used for clinical validation. The primary outcome was overall survival (OS).ResultsBoth assays demonstrated excellent reproducibility. The nanoString assay detected upregulation of the 24-gene signature under hypoxia in vitro, and 16/24 hypoxia genes were upregulated in tumours with high CAIX expression in vivo. Patients with hypoxia-high tumours had worse OS in the Manchester (HR 3.05, 95% CI 1.54–5.19, P = 0.0005) and VORTEX (HR 2.13, 95% CI 1.19–3.77, P = 0.009) cohorts. In the combined cohort, it was independently prognostic for OS (HR 2.24, 95% CI 1.42–3.53, P = 0.00096) and associated with worse local recurrence-free survival (HR 2.17, 95% CI 1.01–4.68, P = 0.04).ConclusionsThis study comprehensively validates a microenvironment classifier befitting FFPE STS biopsies. Future uses include: (1) selecting high-risk patients for perioperative chemotherapy; and (2) biomarker-driven trials of hypoxia-targeted therapies.

Profile of MicroRNAs Associated with Death Due to Disease Progression in Metastatic Papillary Thyroid Carcinoma Patients.

Papillary thyroid carcinoma (PTC) is the most common neoplasm of the endocrine system and has an excellent long-term prognosis, with low rates of distant metastatic disease. Although infrequent, there are cases of deaths directly related to PTC, especially in patients with metastatic disease, and the factors that could be associated with this unfavorable outcome remain a major challenge in clinical practice. Recently, research into genetic factors associated with PTC has gained ground, especially mutations in the TERT promoter and BRAF gene. However, the role of microRNAs remains poorly studied, especially in those patients who have an unfavorable outcome at follow-up. This paper aims to evaluate molecular markers related to the different pathological processes of PTC, as well as the histological characteristics of the neoplasm, and to compare this profile with prognosis and death from the disease using an analysis of patients treated for metastatic disease in a single tertiary cancer center. Evaluation of microRNA expression in paraffin-embedded tumor specimens was carried out by quantitative PCR using the TaqMan® Low Density Array (TLDA) system. Metastatic patients who died from progression of PTC had higher expressions of miR-101-3p, miR-17-5p, and miR-191-5p when compared to patients with stable metastatic disease. These findings are of great importance but should be considered as preliminary because of the small sample.

EICOSATETRAYNOIC ACID REGULATES PRO-FIBROTIC PATHWAYS IN AN INDUCED PLURIPOTENT STEM CELL DERIVED MACROPHAGE:HUMAN INTESTINAL ORGANOID MODEL OF ILEAL CROHN’S DISEASE

Abstract INTRODUCTION Biologics targeting TNF are the mainstay of therapy for children with Crohn’s Disease (CD). However, a subset of patients do not respond, progressing to intestinal fibrosis requiring surgical resection. Prior studies have defined an ileal gene expression module linked to future strictures, and identified small molecules which may reverse this gene signature and suppress fibroblast activation. In the current study we developed a pre-clinical model system and tested a lead candidate, eicosatetraynoic acid (ETYA), a Peroxisome Proliferator-Activated Receptor (PPAR) agonist and arachidonic acid metabolism inhibitor. METHODS Peripheral blood samples were collected from pediatric CD patients and induced pluripotent stem cell (iPSC) lines were generated. iPSC were differentiated into human intestinal organoids (HIOs) and macrophage-like cells. Macrophage:HIO co-cultures were exposed to lipopolysaccharide (LPS) with and without eicosatetraynoic acid (ETYA) pre-treatment. Flow cytometry and cytospin characterized macrophage activation markers and morphology. Co-culture populations were harvested, and RNA and conditioned media were isolated for downstream analysis. TaqMan Low Density Array, Luminex multiplex assay, immunohistologic staining, and sirius red polarized light microscopy were performed to quantify measures of inflammation and fibrosis, and to test whether introduction of ETYA abated any of these inflammatory or fibrotic changes. RESULTS iPSC-derived macrophages exhibited morphology similar to primary macrophages, and expressed inflammatory macrophage cell surface markers including CD68 (Fig. 1A). LPS-stimulated iPSC-derived macrophages expressed a global pattern of gene expression by RNA sequencing enriched in CD ileal inflammatory macrophages (ToppCell Atlas, p=4.397E-117), and produced cytokines and chemokines implicated in refractory disease (Fig. 1B). Co-culture of LPS-primed macrophages with HIO led to up-regulation of the fibroblast activation genes ACTA2 and COL1A1 (Fig. 2). Under these conditions, HIO collagen content measured by sirius red staining and polarized light microscopy was increased (Fig. 2). ETYA pre-treatment prevented these pro-fibrotic effects of LPS-primed macrophages upon HIO gene expression and collagen content. However, LPS induction of macrophage IL1B, TNF, and OSM production was not suppressed by ETYA, suggesting an alternative mechanism of action upon HIO fibroblast activation and collagen content in the co-culture system. CONCLUSIONS ETYA inhibits effects of LPS-primed macrophages upon HIO pro-fibrotic gene expression and collagen production. This was not associated with an effect of ETYA upon macrophage inflammatory cytokine production. Future studies will test alternate pathways including PPAR activation and arachidonic acid metabolism which may mediate this response.

Insights on the pathogenesis of type 2 diabetes as revealed by signature genomic classifiers in an African American population in the Washington, DC area.

African Americans (AA) in the United States have a high risk of type 2 diabetes mellitus (T2DM) and suffer from disparities in the prevalence, mortality, and comorbidities of the disease compared to other Americans. The present study aimed to shed light on the molecular mechanisms of disease pathogenesis of T2DM among AA in the Washington, DC region. We performed TaqMan Low Density Arrays (TLDA) on 24 genes of interest that belong to three categories: metabolic disease and disorders, cancer-related genes, and neurobehavioural disorders genes. The 18 genes, viz. ARNT, CYP2D6, IL6, INSR, RRAD, SLC2A2 (metabolic disease and disorders), APC, BCL2, CSNK1D, MYC, SOD2, TP53 (Cancer-related), APBA1, APBB2, APOC1, APOE, GSK3B, and NAE1 (neurobehavioural disorders), were differentially expressed in T2DM participants compared to controls. Our results suggest that factors including gender, smoking habits, and the severity or lack of control of T2DM (as indicated by HbA1c levels) were significantly associated with differential gene expression. APBA1 was significantly (p-value <0.05) downregulated in all diabetes participants. Upregulation of APOE and CYP2D6 genes and downregulation of the INSR gene were observed in the majority of diabetes patients. Tobacco smoking and gender were significantly associated with case-control differences in expression of the APBA1 and APOE genes (connected with Alzheimer's disease) and the INSR and CYP2D6 (associated with metabolic disorders). The results highlight the need for more effective management of T2DM and for tobacco smoking cessation interventions in this community, and further research on the associations of T2DM with other disease processes, including cancer and neurobehavioral pathways.

Spontaneous head twitches in aged rats: behavioral and molecular study

RationaleWe have discovered that rats at the age of 18 months begin to twitch their heads spontaneously (spontaneous head twitching, SHT). To date, no one has described this phenomenon.ObjectivesThe purpose of this study was to characterize SHT pharmacologically and to assess some possible mechanisms underlying SHT.MethodsWistar male rats were used in the study. Animals at the age of 18 months were qualified as HSHT (SHT ≥ 7/10 min observations) or LSHT (SHT < 7/10 min observations). Quantitative real-time PCR with TaqMan low-density array (TLDA) approach was adopted to assess the mRNA expression of selected genes in rat’s hippocampus.ResultsHSHT rats did not differ from LSHT rats in terms of survival time, general health and behavior, water intake, and spontaneous locomotor activity. 2,5-dimethoxy-4-iodoamphetamine (DOI) at a dose of 2.5 mg/kg increased the SHT in HSHT and LSHT rats, while ketanserin dose-dependently abolished the SHT in the HSHT rats. The SHT was reduced or abolished by olanzapine, clozapine, risperidone, and pimavanserin. All these drugs have strong 5-HT2A receptor–inhibiting properties. Haloperidol and amisulpride, as antipsychotic drugs with a mostly dopaminergic mechanism of action, did not influence SHT. Similarly, escitalopram did not affect SHT. An in-depth gene expression analysis did not reveal significant differences between the HSHT and the LSHT rats.ConclusionsSHT appears in some aging rats (about 50%) and is permanent over time and specific to individuals. The 5-HT2A receptor strongly controls SHT. HSHT animals can be a useful animal model for studying 5-HT2A receptor ligands.

In vivo and in vitro matured bovine oocytes present a distinct pattern of single-cell gene expression.

Oocyte gene expression is a well controlled event that promotes gamete competence to undergo maturation, fertilization, and to support early embryo development, directly affecting reproductive outcomes. Considering that in vivo controlled ovarian stimulation or in vitro maturation (IVM) for the acquisition of mature oocytes has distinct implications for gene expression, we sought to evaluate the effects of these procedures on the expression of competence-related genes in single-cell oocytes. Healthy Nelore cows of reproductive age were synchronized to harvest in vivo matured oocytes; ovaries from slaughtered animals were used to obtain cumulus-oocyte complexes that were in vitro matured. Single-cell gene expression was performed using TaqMan Low-Density Arrays and 42 genes were evaluated. In silico analysis of protein interactions and Gene Ontology (GO) analysis was performed. Reduced gene expression was observed for 24 targets in IVM oocytes when compared with those of in vivo matured oocytes (P < 0.05). Differences ranged from 1.5-fold to 4.8-fold higher in in vivo oocytes and the BMP15 (5.28), GDF9 (6.23), NOBOX (7.25), HSPA8 (7.85) and MSX1 (11.00) showed the greatest fold increases. The strongest score of functional interactions was observed between the CDC20 and CKS2, with the differentially expressed gene CDC20 being the main marker behind GO enrichment. IVM negatively affected the expression of important genes related to oocyte competency, and showed higher expression levels in in vivo matured oocytes. In vivo controlled ovarian stimulation may be a better strategy to achieve proper oocyte competence and increase the success of assisted reproductive technologies.

ALL-061 Delineating the Genomic and Proteomic Landscape of BCR-ABL1-like Acute Lymphoblastic Leukemia Subtype: Bench-to-Bedside Translational Research

Baseline detection of Ph/BCR-ABL1-like-ALL cases is extremely beneficial for hematologists/physicians to design personalized medicine and to treat these patients efficiently. Worldwide, researchers use patented TaqMan low-density array (TLDA) gene expression profiling (GEP) to identify these cases. In developing countries, its detection is very challenging and expensive due to the unavailability of patented TLDA and there is a paucity of literature on the identification and characterization of Ph-like ALL cases. Stepwise identification followed by genomic and proteomic characterization of Ph-like ALL cases. This study was conducted at PGIMER, India (2016-2021). Flowcytometric-immunophenotyping and multiplex RT-PCR was performed to detect four recurrent fusion transcripts (n=1021). Transcriptome GEP was performed on 12 Ph-positive ALL cases. Ph-like-ALL cases identified using nCounter Platform (n=96) and RQ-PCR (n=536) were characterized using FISH, DD-PCR, RQ-PCR and MRD-positivity. Nine HKGs' expression was evaluated using 6 algorithms. Furthermore, we built and validated PHi-RACE classifier (n=629 cases) for its rapid detection. Lastly, we delineated the proteomic landscape of Ph-like ALL cases using LC-MS/MS. We studied 1021 treatment-naive and newly diagnosed B-ALL cases in this study cohort. Multiplex assay revealed 18.66% BCR-ABL1, 3.85% TCF3-PBX1, 5.27% ETV6-RUNX1, and 1.21% KMT2A-AFF1. Using transcriptome GEP, we identified 32.05% Ph-like-ALL cases using NanoString and 26.67% using RQ-PCR. These cases were substantially older and represented higher MRD positivity. In CRLF2 subgroup of Ph-like ALLs, we identified chimeric gene fusions including 10% JAK2 rearrangement, 8.33% CRLF2-IGH and CRLF2-P2RY8 translocation with concomitant R683S mutation. We found 8.33% IKZF1 (Δ4-7) deletion using DD-PCR. Furthermore, we developed an online logistic classifier entitled PHi-RACE (sensitivity=95.20%, specificity=83.70%, cut-off >0.28) for its detection using logistic regression. We have validated PHi-RACE classifier (n=93) and identified 56 Ph-like ALL cases. Six algorithms' findings revealed that EEF2, GAPDH and PGK1 represent the best HKGs for its detection. Lastly, from LC-MS/MS findings, we identified 3079 proteins, spliceosomal mediated mRNA splicing pathway and four miRNAs were significantly expressed in Ph-like ALL cases. The authors created and published the PHi-RACE classifier to detect Ph-like ALL cases, which has direct clinical implications. Further, we have delineated the genomic and proteomic landscape of Ph-like-ALL for the first time from developing countries.

MiR-145, miR-92a and miR-375 Show Differential Expression in Serum from Patients with Diabetic Retinopathies.

Diabetic retinopathies are important disabling conditions. Micro-RNAs (miRNAs) are regulators of gene expression and diseases can change their expression. Our aim was to analyze the expression of miRNAs in serum and vitreous samples from patients with diabetic retinopathies. The following groups and number of individuals were included: proliferative diabetic retinopathy (PDR) (n = 16), diabetic macular edema (DME) (n = 17), and idiopathic epiretinal membrane (IEM) as non-diabetic controls (n = 23). The initial miRNA expression was explored using TaqMan low-density arrays (TLDAs) with subsequent validation through a quantitative polymerase chain reaction (qPCR). Target genes were identified through bioinformatic tools for enrichment analysis. The TLDAs revealed the following miRNAs with differential expression in terms of PDR vs. IEM: miR-320a-3p, miR-92a-3p, and miR-375-3p in the serum, with miR-541-5p and miR-223-5p in the vitreous samples. DME vs IEM: miR-486-5p, miR-145-5p, miR-197-3p, and miR-125b-5p in the serum, and miR-212-3p in vitreous samples. PDR vs. DME: miR-486-5p, miR-100-5p, miR-328-3p, miR-660-5p, and miR-145 in the serum and none in the vitreous samples. Validation was confirmed only for miR-145, miR-92a, and miR-375 in the serum. The relevant enriched pathways for these three validated miRNAs, miR-145, miR-92a, and miR-375 were the vascular endothelial growth factor and its receptor, hepatocyte growth factor receptor, epidermal growth factor, focal adhesion, and phosphoinositide 3-kinase. Our results support the involvement of miRNAs in the pathophysiology of diabetic retinopathies and reinforce their potential as biomarkers or therapeutic resources.

299 Oral difelikefalin improves itch and inflammatory biomarkers in atopic dermatitis subjects with moderate-to-severe pruritus

Pruritus is the most frequent and burdensome atopic dermatitis (AD) symptom and can exacerbate disease via the itch-scratch cycle. Difelikefalin (DFK), a kappa-opioid receptor (KOR) agonist is approved for moderate-to-severe pruritus in adults with CKD undergoing hemodialysis. DFK blocks pruritic signaling through peripheral sensory neurons and may have anti-inflammatory activity. We tested the impact of DFK on pruritus-related and inflammatory transcriptomes of AD patients. In a phase 2 clinical study, subjects with AD and moderate-to-severe pruritus were randomized (1:1:1:1) to receive oral twice-daily DFK (3 doses) or placebo for 12 wk. A substudy (n=40) characterized effects of DFK on pruritus- and AD-related gene expression using baseline and wk 12 skin biopsies. Gene expression was measured using RNA-sequencing and TaqMan Low-Density Array qualitative polymerase chain reaction. Data from DFK groups were pooled. DFK altered expression of multiple individual pruritus- and AD-related genes. Gene set variation analysis confirmed downregulation of pruritus-related genes (eg, IL-31, TRPV2) and the Th2 pathway following 12 wk treatment with DFK, but not placebo. Changes in gene expression of pruritus- and immune-related genes significantly correlated with skin improvement for DFK-treated subjects. DFK downregulated expression of key genes implicated in pruritus and AD inflammation. Reports demonstrate an anti-pruritic effect of DFK in AD and other chronic itch conditions. DFK is a promising therapy for AD-related pruritus and may provide anti-inflammatory benefit by impacting the itch-scratch cycle.