Blood mRNA Research Articles

Exercise Augments the Effect of SGLT2 Inhibitor Dapagliflozin on Experimentally Induced Diabetic Cardiomyopathy, Possible Underlying Mechanisms.

One of the most prevalent cardiovascular problems linked with type 2 diabetes mellitus (T2DM) is diabetic cardiomyopathy (DCM). DCM is associated with myocardial oxidative stress, inflammation, apoptosis, suppressed autophagy, extracellular matrix remodeling, and fibrosis. The current study aims to investigate the protective effect of sodium-glucose transport 2 inhibitor (SGLT2i) dapagliflozin and/or exercise on DCM. Thirty adult male Sprague Dawley rats are used. T2DM is induced by a 6-week high-fat diet (HFD) followed by a single intraperitoneal (IP) injection of 35 mg/kg streptozotocin (STZ). Rats are divided into five groups, control, diabetic (DM), DM + swimming, DM + dapagliflozin, and DM + dapagliflozin and swimming. Serum glucose, insulin, insulin resistance (HOMA-IR), and cardiac enzymes (CK-MB and lactate dehydrogenase (LDH) are measured. Heart specimens are used for evaluation of cellular oxidative stress markers malondialdehyde (MDA), antioxidant enzymes, glutathione (GSH), and catalase (CAT), as well as mRNA expression of TGF-β, MMP9, IL-1β, and TNF-α. Stained sections with haematoxylin and eosin (H & E) and Masson trichrome are used for histopathological evaluation and detection of fibrosis, respectively. Immunohistochemical staining for apoptosis (caspase-3), and autophagy (LC3) are also carried out. The combinations of SGLT2i and exercise exhibited the most significant cardioprotective effect. It improved diabetic-induced histopathological alterations in the myocardium and attenuated the elevation of serum blood glucose, CK-MB, LDH, myocardial MDA, and mRNA expression of TNF-α, IL-1β, TGF-β, MMP9, and the immune expression of caspase-3. Moreover, this combination increased the serum insulin, myocardial antioxidants GSH and CAT, and increase the immune expression of the LC-3. In conclusion, a combination of SGLT2i and exercise exerted a better antioxidant, anti-inflammatory, and antifibrotic effect in DCM. Moreover, the combination enhances the autophagic capacity of the heart.

CircRNA, lncRNA, and mRNA profiles of umbilical cord blood exosomes from preterm newborns showing bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) represents a multifactorial chronic pulmonary pathology and a major factor causing premature illness and death. The therapeutic role of exosomes in BPD has been feverishly investigated. Meanwhile, the potential roles of exosomal circRNAs, lncRNAs, and mRNAs in umbilical cord blood (UCB) serum have not been studied. This study aimed to detect the expression profiles of circRNAs, lncRNAs, and mRNAs in UCB-derived exosomes of infants with BPD. Microarray analysis was performed to compare the RNA profiles of UCB-derived exosomes of a preterm newborn with (BPD group) and without (non-BPD, NBPD group) BPD. Then, circRNA/lncRNA–miRNA–mRNA co-expression networks were built to determine their association with BPD. In addition, cell counting kit-8 (CCK-8) assay was used to evaluate the proliferation of lipopolysaccharide (LPS)-induced human bronchial epithelial cells (BEAS-2B cells) and human umbilical vein endothelial cells (HUVECs). The levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in LPS-induced BEAS-2B cells and HUVECs were assessed through Western blot analysis. Then, quantitative reverse transcription–polymerase chain reaction assay was used to evaluate the expression levels of four differentially expressed circRNAs (hsa_circ_0086913, hsa_circ_0049170, hsa_circ_0087059, and hsa_circ_0065188) and two lncRNAs (small nucleolar RNA host gene 20 (SNHG20) and LINC00582) detected in LPS-induced BEAS-2B cells or HUVECs. A total of 317 circRNAs, 104 lncRNAs, and 135 mRNAs showed significant differential expression in UCB-derived exosomes of preterm infants with BPD compared with those with NBPD. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to examine differentially expressed exosomal circRNAs, lncRNAs, and mRNAs. The results showed that the GO terms and KEGG pathways mostly involving differentially expressed exosomal RNAs were closely associated with endothelial or epithelial cell development. In vitro, CCK-8 and Western blot assays revealed that LPS remarkably inhibited the viability and promoted inflammatory responses (TNF-α and IL-1β) of BEAS-2B cells or HUVECs. The expression levels of circRNAs hsa_circ_0049170 and hsa_circ_0087059 were upregulated in LPS-induced BEAS-2B cells; the expression level of hsa_circ_0086913 was upregulated and that of hsa_circ_0065188 was downregulated in LPS-induced HUVECs. Moreover, the expression level of lncRNA SNHG20 was upregulated and that of LINC00582 was downregulated in LPS-induced BEAS-2B cells. Further, 455 circRNA/lncRNA–miRNA–mRNA interaction networks were predicted, including hsa_circ_0086913/hsa-miR-103a-3p/transmembrane 4 L six family member 1 (TM4SF1) and lncRNA-SNHG20/hsa-miR-6720-5p/spermine synthase (SMS) networks, which may take part in BPD.Conclusion: This study provided a systematic perspective on UCB-derived exosomal circRNAs and lncRNAs and laid an important foundation for further investigating the potential biological functions of exosomal circRNAs and lncRNAs in BPD.What is Known:• BPD represents a multifactorial chronic pulmonary pathology and a major factor causing premature illness and death.• The therapeutic role of exosomes in BPD has been feverishly investigated, and exosomal RNAs were ignored.What is New:• The profiles of UCB-derived exosomal circRNAs, lncRNAs, and mRNAs were performed.• Several differentially expressed circRNAs and lncRNAs were identified in LPS-induced BEAS-2B cells and HUVECs.

Vaccine mRNA Can Be Detected in Blood at 15 Days Post-Vaccination.

COVID-19 mRNA vaccines effectively reduce incidence of severe disease, hospitalisation and death. The biodistribution and pharmacokinetics of the mRNA-containing lipid nanoparticles (LNPs) in these vaccines are unknown in humans. In this study, we used qPCR to track circulating mRNA in blood at different time-points after BNT162b2 vaccination in a small cohort of healthy individuals. We found that vaccine-associated synthetic mRNA persists in systemic circulation for at least 2 weeks. Furthermore, we used transmission electron microscopy (TEM) to investigate SARS-CoV-2 spike protein expression in human leukemic cells and in primary mononuclear blood cells treated in vitro with the BNT162b2 vaccine. TEM revealed morphological changes suggestive of LNP uptake, but only a small fraction of K562 leukemic cells presented spike-like structures at the cell surface, suggesting reduced levels of expression for these specific phenotypes.

Suppressors of Cytokine Signaling Are Decreased in Major Depressive Disorder Patients.

There is strong evidence for an association between major depressive disorder (MDD) and inflammation. However, some studies have not observed an increase in inflammatory cytokines in MDD, and the mechanism behind this is unknown. In the present study, we evaluated MDD severity using the Montgomery–Åsberg Depression Rating Scale (MADRS) and quantified mRNA levels of the blood inflammatory cytokines interleukin (IL) 1β, IL-6 and tumor necrosis factor alpha (TNF-α), as well as negative regulators of cytokine signaling—comprising IL-10, IL-1RA, SOCS1, SOCS2 and SOCS3—in MDD patients (n = 36), with a focus on mild MDD, and normal controls (NC, n = 30). We also measured the serum levels of IL-1β and IL-6. Neither the blood mRNA nor the protein levels of inflammatory cytokines were significantly elevated in the MDD group compared with the NC group. However, we observed significant decreases in SOCS1, SOCS2 and SOCS3 mRNA in the MDD group compared to the NC group. A significant finding was a decrease in SOCS3 mRNA after remission from MDD, suggesting that SOCS3 is a trait marker in depressive symptoms. We consider that our findings would be useful in elucidating the pathophysiological mechanism of depression.

LINC00461 Regulates the Recurrence of Large B Cell Lymphoma through the miR-411-5p/BNIP3 Pathway.

Objective To analyze the mechanism of LINC00461 regulating the recurrence of diffuse large B cell lymphoma (DLBCL) through microRNA (miR)-411-5p/BCL2 interacting protein 3 (BNIP3) pathway. Methods DLBCL samples in TCGA and GSE12453 were used for differential analysis to find long noncoding RNA (lncRNA) related to DLBCL recurrence. The 4 DLBCL data with the highest and lowest expression levels of LINC00461 in the TCGA database were selected for GSEA enrichment analysis. The targeting relationships of miR-411-5p with LINC00461 and BNIP3 were verified by the dual luciferase report. Blood samples from DLBCL patients were used to analyze the correlation between miR-411-5p and LINC00461 or BNIP3. LINC00461, miR-411-5p, or BNIP3 was overexpressed or silenced by transfection, and a tumor-bearing nude mice model was constructed to detect their effects on proliferation and apoptosis. Results The level of LINC00461 in DLBCL was significantly higher than that in normal cases, and the level in recurrence DLBCL was significantly higher than that in nonrecurrence. The enrichment analysis results showed that the function of LINC00461 was closely related to apoptosis. The results shown that miR-411-5p bound to LINC00461 and BNIP3 and was negatively correlated with LINC00461 and BNIP3 mRNA in blood of DLBCL patients. Suppressing the level of LINC00461 inhibited cell proliferation and induced apoptosis. The inhibition of LINC00461 or overexpression of miR-411-5p reduced the expression of BNIP3 protein, thereby inducing apoptosis at the in vivo and in vitro levels. Conclusion LINC00461 may induce miR-411-5p to “sponge,” thereby increasing the expression of BNIP3 protein, and exerting the function of inhibiting apoptosis and promoting DLBCL recurrence.

P790: THE EXPRESSION LEVEL OF WT1 MRNA IN PATIENTS WITH IDIOPATHIC CYTOPENIA OF UNDETERMINED SIGNIFICANCE (ICUS) PROGRESSING TO MYELODYSPLASTIC SYNDROME (MDS)

Background: Idiopathic cytopenia of undetermined significance (ICUS) is defined as patients with one or more unexplained cytopenia who do not meet diagnostic criteria for myelodysplastic syndrome (MDS) or another hematologic disorder. In the clinical course of ICUS, some patients eventually have typical MDS changes. Until now, there is no effective method to predict the development of the disease. Recent studies have shown that the expression level of WT1 mRNA in peripheral blood has important clinical significance in the diagnosis, occurrence, development and prognosis of MDS. At the same time, some studies have proved that there is a significant difference in the expression level of WT1 mRNA between MDS and hemocytopenia caused by other reasons. Aims: In order to clarify the clinical significance of WT1 mRNA expression in the progression of ICUS to MDS. Methods: Between April 2017 and January 2020, untreated adult patients with ICUS or lower-risk MDS were enrolled in the study. All patients detected the WT1 mRNA expression of bone marrow and peripheral blood at diagnosis. Then patients with ICUS were followed up. Every three months, the patients with ICUS detected the WT1 mRNA expression of peripheral blood, until the patient progressed to MDS. The results of WT1 transcript levels are depicted as WT1 (copies/mL)/c-ABL (copies/mL) as a percentage. Results: Total of 80 patients with ICUS and 40 patients with lower-risk MDS were enrolled. At diagnosis, the median expression level of WT1 mRNA in MDS group was 5.99 (0.08-79.27), and that in ICUS group was 0.50 (0.16-22.48). There was significant difference between them (P = 0.000). Then 80 patients with ICUS were followed up to January 2022. The median follow-up time was 30 months (11-54 months). 8 patients developed MDS, the progression rate was 10%, and the median progression time was 6.5 months (2-19 months). The median expression level of WT1 mRNA at the initial diagnosis in ICUS group without progressing to MDS was 0.44 (0.16-7.58), and that in progressing to MDS group was 9.44 (0.26-22.48). There was significant difference between the two groups (P = 0.031). The median expression level of WT1 mRNA was 19.185 (0.73-42.30) when MDS was diagnosed. The median maximum expression level of WT1 mRNA in peripheral blood of ICUS group without MDS was 0.98 (0.22-7.58). There was significant difference between the two groups (P = 0.007). Summary/Conclusion: The study clarified the WT1 mRNA expression status of ICUS patients. There were significant differences in WT1 mRNA expression level between patients with ICUS and lower-risk MDS, and the WT1 mRNA expression level of patients with lower-risk MDS was relatively high. The study confirmed that patients with high WT1 mRNA expression level in the patients with ICUS (WT1 mRNA expression level > 9.44) are very likely to progressing to MDS; Dynamic monitoring of WT1 mRNA expression in peripheral blood is an effective method for following-up after ICUS diagnosis. The progressive increase of WT1 mRNA expression is one of the indicators for researchers to judge the progress of ICUS to MDS.

Soluble PD-L1 in blood correlates positively with neutrophil and negatively with lymphocyte mRNA markers and implies adverse sepsis outcome

Sepsis causes a myriad of immunological reactions that result in life-threatening alterations in the human body. Immunosuppression in sepsis is partly attributed to the programmed death receptor (PD-1) and its associated ligand (PD-L1) via the regulation of lymphocytes and neutrophils. Although the soluble forms of these proteins (i.e., sPD-1 and sPD-L1, respectively) are recognized as possible sepsis biomarkers, their functional implications are yet to be elucidated. Our research assessed the correlation between sPD-1 and sPD-L1 and blood mRNA markers and sepsis outcome. Blood samples of septic patients of urogenital origin versus control patients (both groups: n = 18) were analyzed. Blood serum sPD-1 and sPD-L1 levels were determined using the enzyme-linked immunosorbent assay (ELISA). The whole blood mRNA concentrations of PD-1, PD-L1, neutrophil markers (CEACAM8 and MPO), and T-lymphocyte markers (TCRβ, CD4 and CD8) were determined via reverse transcriptase quantitative PCR (RT-qPCR). sPD-L1 levels were significantly increased in septic patients when compared to the controls, whereas sPD-1 levels were unaltered. Patients with high sPD-L1 levels, as dichotomized to the median, had a significantly shorter survival rate than those with low sPD-L1 levels. The sensitivity/specificity characteristics of sPD-L1 proved significant for sepsis detection. Furthermore, sPD-L1 correlated with the mRNA concentrations of PD-L1, CEACAM, and MPO, as well as major inflammatory markers (C-reactive protein and procalcitonin). However, sPD-L1 negatively correlated with TCRβ, CD4, and CD8 mRNAs. sPD-L1 was found to be significantly increased in septic patients. Notably, sPD-L1 correlated with PD-L1 mRNA and neutrophil markers and was indicative of adverse outcomes.

Association of NKAPL rs1635 With Cognitive Function in Early-Onset Schizophrenia

BACKGROUND: Schizophrenia is a severe mental disorder with high heritability, and cognitive dysfunction is one of the core features. Growing evidence suggests the genetic risk of schizophrenia may contribute to cognitive impairments. The variant rs1635 (nucleotide sequence: c.455C>A; amino acid sequence: T152N) located on the (NFKB activating protein like) NKAPL gene confers risk for schizophrenia and might play a role in the neurodevelopmental process, which is particularly relevant to cognitive function. However, the relationship between rs1635 and cognitive function remains unclear. METHODS: A total of 130 patients with early-onset schizophrenia (EOS) and 300 patients with adult-onset schizophrenia (AOS) of Han Chinese were recruited and underwent neurocognitive tests by using the MATRICS Consensus Cognitive Battery (MCCB). The NKAPL rs1635 was genotyped by using DNA sequencing. The peripheral blood NKAPL mRNA expression level was examined in 152T or 152N carriers (n = 20) in EOS patients, by using the qRT-PCR. The phosphorylation level of NAKPL T152N polymorphism was detected by cell experiments. In utero electroporation of mouse embryos was examined to explore the effect of Nkapl on neuronal migration. RESULTS: Compared with rs1635 AA and AC carriers, CC (the CC genotype encodes the protein NKAPL-152T) carriers of EOS patients performed better in cognitive domain of speed of processing (t = 2.644, p = 0.009), trail making test (t = 2.221, p = 0.028) and category fluency (t = 2.578, p = 0.011). However, patients with AOS exhibited no significant differences in seven domains among the three genotype groups. There were no significant differences in cognitive performance between EOS and AOS. In EOS patients, NKAPL mRNA level in NKAPL-152N carriers is significantly lower than that of NKAPL-152T carriers. The phosphorylation level of NKAPL-152N is significantly decreased compared to NKAPL-152T. In utero electroporation showed that Nkapl deletion impairs the embryonic radial migration process. CONCLUSION: The present study found that NKAPL rs1635 was associated with cognitive impairments and peripheral blood mRNA expression level in EOS patients. The NKAPL full-length protein is required for embryonic cortical neuronal migration. The phosphorylation level of NKAPL-152N is significantly decreased. The NKAPL T152N may affect the NAKPL mRNA expression level and embryonic cortical neuronal migration by regulating the NAKPL protein phosphorylation. These data suggest that NKAPL rs1635 affects cognitive function by regulating early brain development in early-onset schizophrenia.

Abstract CT011: NeoCOAST: open-label, randomized, phase 2, multidrug platform study of neoadjuvant durvalumab alone or combined with novel agents in patients (pts) with resectable, early-stage non-small-cell lung cancer (NSCLC)

Abstract Background: Neoadjuvant anti-PD-(L)1 therapy may offer clinical benefit in resectable, early-stage NSCLC. NeoCOAST (NCT03794544) is a global, randomized phase 2 study of the anti-PD-L1 monoclonal antibody (mAb) durvalumab (D) alone or combined with the anti-CD73 mAb oleclumab (O), the anti-NKG2A mAb monalizumab (M), or the anti-STAT3 antisense oligonucleotide danvatirsen (Da) as neoadjuvant therapy. Methods: Pts with previously untreated, cytologically/histologically documented, resectable, Stage I [>2 cm]-IIIA NSCLC and ECOG PS 0-1 were randomized 1:1:1:1 (stratified by lymph node involvement) to receive D 1500 mg IV alone Q4W or combined with O 3000 mg IV Q2W, M 750 mg IV Q2W, or Da 200 mg IV QW, for one 28-day cycle, followed by surgery. Da monotherapy was also given on days 1, 3 and 5 of the week prior to combination treatment. The primary endpoint was investigator-assessed major pathological response (MPR; ≤10% residual viable tumor cells at tumor site and nodes, at surgery). Secondary endpoints were pathological complete response (pCR; no viable tumor cells), safety and tolerability, feasibility of surgery, PK and immunogenicity. Exploratory endpoints included tumor and microbiome biomarkers and blood mRNA signatures. Results: From March 2019 to Sept 2020, 84 pts were randomized and 83 received D (n=26), D+O (n=21), D+M (n=20), or D+Da (n=16). MPR occurred in 11.1% (95% CI, 2.4-29.2), 19.0% (95% CI, 5.4-41.9), 30.0% (95% CI, 11.9-54.3), and 31.3% (95% CI, 11.0-58.7%), respectively. pCR occurred in 3.7% (95% CI, 0.1-19.0), 9.5% (95% CI, 1.2-30.4), 10.0% (95% CI, 1.2-31.7%) and 12.5% (95% CI, 1.6-38.3), respectively. Rates of treatment-related AEs were 34.6% with D, 57.1% with D+O, 50.0% with D+M, and 43.8% with D+Da (grade ≥3 in 0%, 4.8%, 0% and 6.3%, respectively). Most pts (76/83; 91.6%) completed surgery with no significant delay; of the 7 pts unable to, 5 had progressive or stage IV disease. Across all arms, MPR was more common in pts with baseline tumor PD-L1 expression ≥1% vs those with <1%. With D, CD73 expression was correlated with greater residual viable tumor cells at surgery; however, with D+O, high CD73 (≥10% tumor cells) was associated with reduced viable tumor cells. Transcriptome analysis of post-treatment and baseline blood samples showed upregulation of genes involved in B cell activation and antigen presentation in pts with MPR in the D+O arm, and upregulation of genes associated with Tregs in pts with MPR in the D+M arm. Conclusions: One cycle of D + O, M or Da improved MPR and pCR rates vs D alone, with no new safety signals. Responses were associated with baseline tumor PD-L1 and CD73 expression levels. Pts with MPR receiving D+O or D+M had peripheral transcriptomic signatures related to immune cell function. These data warrant further investigation of these agents in resectable NSCLC. Citation Format: Tina Cascone, Rosario García-Campelo, Jonathan Spicer, Walter Weder, Davey Daniel, David Spigel, Maen Hussein, Julien Mazieres, Julio Oliveira, Edwin Yau, Alexander Spira, Raymond Mager, Oday Hamid, Lin-Yang Cheng, Ying Zheng, Jorge Blando, Lara McGrath, Italia Grenga, Yee Soo-Hoo, Rakesh Kumar, Patrick Forde. NeoCOAST: open-label, randomized, phase 2, multidrug platform study of neoadjuvant durvalumab alone or combined with novel agents in patients (pts) with resectable, early-stage non-small-cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT011.

Metals and Metallothionein Expression in Relation to Progression of Chronic Kidney Disease of Unknown Etiology (CKDu) in Sri Lanka.

Chronic kidney disease of unknown etiology was investigated for metal relations in an endemic area by a cross-sectional study with CKD stages G1, G2, G3a, G3b, G4, G5 (ESRD), and endemic and nonendemic controls (EC and NEC) as groups. Subjects with the medical diagnosis were classified into groups by eGFR (SCr, CKD-EPI) and UACR of the study. It determined 24 metals/metalloids in plasma (ICPMS) and metallothionein (MT) mRNA in blood (RT-PCR). MT1A at G3b and MT2A throughout G2–G5 showed increased transcription compared to NEC (ANOVA, p < 0.01). Both MT1A and MT2A remained metal-responsive as associations emerged between MT2A and human MT inducer Cr (in EC: r = 0.54, p < 0.05, n = 14), and between MT1A and MT2A (in EC pooled with G1–G5: r = 0.58, p < 0.001, n = 110). Human MT (hMT)-inducers, namely Zn, Cu, As, Pb, and Ni; Σ hMT-inducers; 14 more non-inducer metals; and Σ MT-binding metals remained higher (p < 0.05) in EC as compared to NEC. Declining eGFR or CKD progression increased the burden of Be, Mg, Al, V, Co, Ni, Rb, Cs, Ba, Mn, Zn, Sr, Σ hMT-inducers, and Σ MT-binding metals in plasma, suggesting an MT role in the disease. MT1A/2A mRNA followed UACR (PCA, Dendrogram: similarity, 57.7%). The study provides evidence that proteinuric chronic renal failure may increase plasma metal levels where blood MT2A could be a marker.

Predicting outcomes with circulating adrenergic neuroblastoma mRNAs in children with relapsed and refractory neuroblastoma: A BEACON-Neuroblastoma biomarker study.

10039 Background: Children with relapsed and refractory neuroblastoma (RR-NBL) have poor outcomes. Early identification of children at greatest risk of relapse could mean timelier modifications of treatment to improve outcomes. High levels of adrenergic neuroblastoma mRNAs in blood of children with stage M neuroblastoma receiving frontline treatment predict poor outcome (Viprey PMID: 24590653). Since these markers have not been thoroughly studied in the RR-NBL population, we have prospectively evaluated the prognostic potential of the adrenergic neuroblastoma mRNAs paired-like homeobox 2B (PHOX2B ) and tyrosine hydroxylase (TH) in blood from children with RR-NBL treated in the BEACON-Neuroblastoma trial (NCT02308527). Methods: Blood samples collected at baseline from 88 children were analysed by reverse transcriptase polymerase chain reaction (RTqPCR) for PHOX2B and TH mRNAs. The prognostic power of these mRNAs was evaluated using Kaplan-Meier survival curves and Cox proportional hazards regression. Progression-free (PFS) and overall survival (OS) were calculated from the date that the blood sample was taken at screening to the date of an event; progression, disease recurrence, death or censored alive at the last clinical evaluation. Results: Of the children in this cohort, 58 (66%) had relapsed and 30 (34%) had refractory disease. Twenty-three (26%) had MYC-N amplified tumours. TH and PHOX2B mRNAs were detected in 55% and 60% of blood samples respectively; the correlation coefficient between TH and PHOX2B was 0.75. Higher levels of TH, PHOX2B mRNAs or both combined were associated with reduced PFS and OS (Table). For TH, median PFS for children with TH levels below the median was 12 months (95%CI, 4.6–13 months) versus 5.5 months (95%CI, 1.8–9.4 months) for those children with TH levels above the median. For PHOX2B, median PFS for children with PHOX2B levels below the median was 11.5 months (95%CI, 7.6–34 months), compared to 5.7 months (95%CI, 1.8–10.5 months) where levels were above the median. Conclusions: TH and PHOX2B mRNAs in blood collected at baseline identify children with refractory or relapsed neuroblastoma at greatest risk of progression or death. In the RR-NBL setting, this simple blood test could be used to stratify treatment strategies. Clinical trial information: NCT02308527. [Table: see text]

Association Study and Meta-Analysis of Polymorphisms and Blood mRNA Expression of the ALDH2 Gene in Patients with Alzheimer’s Disease

Background:Late-onset Alzheimer’s disease (LOAD) is a complex disease in which neuroinflammation plays an important pathophysiological role, and exposure to neurotoxic substrates such as aldehydes may contribute. Blood mRNA expression levels of neuroinflammation-related genes appear to be potential biological markers of LOAD. A relationship between ALDH2 and LOAD has been suggested.Objective:Our objective was to examine blood ALDH2 expression in Japanese LOAD patients, conduct a genetic association study, and add new studies to an extended meta-analysis of the Asian population.Methods:A blood expression study (45 AD subjects, 54 controls) in which total RNA was isolated from whole peripheral blood samples and ALDH2 expression measured was conducted. In addition, a genetic association study (271 AD subjects, 492 controls) using genomic DNA from whole peripheral blood samples was conducted. Finally, a meta-analysis examined the relationship between ALDH2*2 frequency and the risk of LOAD.Results:ALDH2 mRNA expression was significantly higher in LOAD than in controls, and also higher in men with LOAD than in women with LOAD (p = 0.043). The genotypes in the two classified groups and the allele frequency were significantly different between AD and control subjects. The meta-analysis showed a significant difference in the ALDH2*2 allele, with an increased AD risk (OR = 1.38; 95% CI = 1.02–1.85; p = 0.0348, I2 = 81.1%).Conclusion:There was a significant increase in blood ALDH2 expression, and a genetic association with ALDH2*2 in LOAD. ALDH2 may have significant roles in the pathogenesis of LOAD in the Asian population.

Diagnosis of pulmonary tuberculosis via identification of core genes and pathways utilizing blood transcriptional signatures: a multicohort analysis

BackgroundBlood transcriptomics can be used for confirmation of tuberculosis diagnosis or sputumless triage, and a comparison of their practical diagnostic accuracy is needed to assess their usefulness. In this study, we investigated potential biomarkers to improve our understanding of the pathogenesis of active pulmonary tuberculosis (PTB) using bioinformatics methods.MethodsDifferentially expressed genes (DEGs) were analyzed between PTB and healthy controls (HCs) based on two microarray datasets. Pathways and functional annotation of DEGs were identified and ten hub genes were selected. They were further analyzed and selected, then verified with an independent sample set. Finally, their diagnostic power was further evaluated between PTB and HCs or other diseases.Results62 DEGs mostly related to type I IFN pathway, IFN-γ-mediated pathway, etc. in GO term and immune process, and especially RIG-I-like receptor pathway were acquired. Among them, OAS1, IFIT1 and IFIT3 were upregulated and were the main risk factors for predicting PTB, with adjusted risk ratios of 1.36, 3.10, and 1.32, respectively. These results further verified that peripheral blood mRNA expression levels of OAS1, IFIT1 and IFIT3 were significantly higher in PTB patients than HCs (all P < 0.01). The performance of a combination of these three genes (three-gene set) had exceeded that of all pairwise combinations of them in discriminating TB from HCs, with mean AUC reaching as high as 0.975 with a sensitivity of 94.4% and a specificity of 100%. The good discernibility capacity was evaluated d via 7 independent datasets with an AUC of 0.902, as well as mean sensitivity of 87.9% and mean specificity of 90.2%. In regards to discriminating PTB from other diseases (i.e., initially considered to be possible TB, but rejected in differential diagnosis), the three-gene set equally exhibited an overall strong ability to separate PTB from other diseases with an AUC of 0.999 (sensitivity: 99.0%; specificity: 100%) in the training set, and 0.974 with a sensitivity of 96.4% and a specificity of 98.6% in the test set.ConclusionThe described commonalities and unique signatures in the blood profiles of PTB and the other control samples have considerable implications for PTB biosignature design and future diagnosis, and provide insights into the biological processes underlying PTB.

Effects of Nigella sativa supplementation on blood concentration and mRNA expression of TNF-α, PPAR-γ and adiponectin, as major adipogenesis-related markers, in obese and overweight women: a crossover, randomised-controlled trial.

Adipocyte expansion through adipogenesis can offset the adverse metabolic effects of obesity. Nigella sativa (NS) (black seed) oil is shown to have therapeutic features in the management of obesity. NS oil might have beneficial changes in obese populations through mediating serum levels of adipogenesis-related parameters and relative transcriptional gene-diet interactions (nutrigenomics), though no previous studies assessed this mechanism in overweight/obese participants. This study assessed the effects of NS oil supplements on blood concentration and mRNA expression levels of TNF-α, PPAR-γ and serum adiponectin and expression of AdipoR1, as major adipogenesis and obesity-related parameters, in overweight/obese women using a cross-over design. Eligible women were randomised to receive either NS oil supplements (2000 mg/d) or placebo. Two periods of interventions (8 weeks in each) were cross-changed by a 4-week washout period. An individualised diet plan without calorie deficits was given to participants to match their energy/macronutrient intakes. The Pkcross procedure and intention-to-treat analysis were performed using Stata. Cohen's d(d) was estimated to measure the magnitude of the effects. Forty-six participants were included. NS oil capsules reduced transcription levels ((d = -2·31), P < 0·001) and blood concentrations of TNF-α ((d = -0·29), P < 0·001). AdipoR1 expression (d = 2·24, P < 0·001) and serum adiponectin (d = 0·88, P < 0·001) showed a significant augmentation with a medium-high effect size, as did gene expression (d = 0·69, P < 0·001) and serum levels of PPAR-γ (d = 0·97, P < 0·001). There was a moderate but significant decrease in body weight (d = 0·6, P < 0·001). The present beneficial findings would provide strong information for future nutrigenomics/clinical trial studies assessing the role of NS in the management of obesity and other comorbidities.

MO943: Levels of Forkhead Box P3 (FOXP3) Transcripts in Kidney Transplant Recipients is Associated With Post-Transplant Cytomegalovirus Viremia

Abstract BACKGROUND AND AIMS The forkhead box P3 (FOXP3) transcription factor is essential for the differentiation of T regulatory cells, which are major regulators of immune tolerance and immunologic activity. Quantification of the FOXP3 mRNA in peripheral blood might be clinically useful. Considering possible post-transcriptional regulation of FOXP3 mRNA, we tested the hypothesis that a low level of FOXP3 is associated with a higher risk of cytomegalovirus (CMV) viremia, in kidney transplant recipients (KTRs). METHOD Blood samples were collected the first day after transplantation from incident adult kidney transplant recipients. Ribonucleic acid was extracted from peripheral blood mononuclear cells and used in quantitative reverse transcription polymerase chain reaction. Levels of FOXP3 were measured using two primers differentiating mRNA FOXP3 and pre-mRNA FOXP3 (pre-FOXP3), both primers were normalized to the reference gene β-actin. We defined viremia with CMV as the finding of more than 1000 copies per mL of CMV DNA in plasma, at least once, within 3 months after transplantation. A ratio of FOXP3 to pre-FOXP3 was calculated and compared, using Mann–Whitney U-test according to CMV viremia status. Finally using receiver operating characteristics, we calculated are under the curve. RESULTS Of 340 patients enrolled, 24 experienced post-transplant CMV viremia. The median ratio of FOXP3 to pre-FOXP3 was lower in those diagnosed with post-transplant CMV viremia, compared to those who were not 6.8 [1.2–11.3] versus 12.2 [3.5–28.9] P &amp;lt; 0.01, respectively. Receiver operating characteristics yielded an area under the curve of 66.7% P &amp;lt; 0.01. CONCLUSION Our data shows an association between low levels of FOXP3 and risk for post-transplant CMV infection in kidney transplant recipients.

Gene expression changes implicate specific peripheral immune responses to Deep and Lobar Intracerebral Hemorrhages in humans

The peripheral immune system response to Intracerebral Hemorrhage (ICH) may differ with ICH in different brain locations. Thus, we investigated peripheral blood mRNA expression of Deep ICH, Lobar ICH, and vascular risk factor-matched control subjects (n = 59). Deep ICH subjects usually had hypertension. Some Lobar ICH subjects had cerebral amyloid angiopathy (CAA). Genes and gene networks in Deep ICH and Lobar ICH were compared to controls. We found 774 differentially expressed genes (DEGs) and 2 co-expressed gene modules associated with Deep ICH, and 441 DEGs and 5 modules associated with Lobar ICH. Pathway enrichment showed some common immune/inflammatory responses between locations including Autophagy, T Cell Receptor, Inflammasome, and Neuroinflammation Signaling. Th2, Interferon, GP6, and BEX2 Signaling were unique to Deep ICH. Necroptosis Signaling, Protein Ubiquitination, Amyloid Processing, and various RNA Processing terms were unique to Lobar ICH. Finding amyloid processing pathways in blood of Lobar ICH patients suggests peripheral immune cells may participate in processes leading to perivascular/vascular amyloid in CAA vessels and/or are involved in its removal. This study identifies distinct peripheral blood transcriptome architectures in Deep and Lobar ICH, emphasizes the need for considering location in ICH studies/clinical trials, and presents potential location-specific treatment targets.

Sulfiredoxin-1 blood mRNA expression levels negatively correlate with hippocampal atrophy and cognitive decline.

Introduction: Cognitive decline, correlating with hippocampal atrophy, characterizes several neurodegenerative disorders having a background of low-level chronic inflammation and oxidative stress. Methods: In this cross-sectional study, we examined how cognitive decline and hippocampal subfields volume are associated with the expression of redox and inflammatory genes in peripheral blood. We analyzed 34 individuals with different cognitive scores according to Mini-Mental State Examination, corrected by age and education (adjMMSE). We identified a group presenting cognitive decline (CD) with adjMMSE<27 (n=14) and a normal cognition (NC) group with adjMMSE≥27 (n=20). A multiparametric approach, comprising structural magnetic resonance imaging measurement of different hippocampal segments and blood mRNA expression of redox and inflammatory genes was applied. Results: Our findings indicate that hippocampal segment volumes correlate positively with adjMMSE and negatively with the blood transcript levels of 19 genes, mostly redox genes correlating especially with the left subiculum and presubiculum. A strong negative correlation between hippocampal subfields atrophy and Sulfiredoxin-1 ( SRXN1) redox gene was emphasized. Conclusions: Concluding, these results suggest that SRXN1 might be a valuable candidate blood biomarker for non-invasively monitoring the evolution of hippocampal atrophy in CD patients.

Abnormal pattern of vitamin D receptor-associated genes and lncRNAs in patients with bipolar disorder

BackgroundBipolar disorder (BD) is a multifactorial condition. Several signaling pathways affect development of this disorder. With the purpose of exploring the role of vitamin D receptor (VDR) signaling in this disorder, we measured expression of selected mRNA coding genes and long non-coding RNAs (lncRNAs) in this pathway in patients versus normal subjects.MethodsWe measured expression of VDR-associated lncRNAs and mRNAs (SNHG6, MALAT1, Linc00511, Linc00346, VDR and CYP27B1) in the peripheral blood of BD patients vs. healthy individuals.ResultsExpression of SNHG6 was significantly higher in cases vs. controls (Posterior beta = 1.29, P value < 0.0001. Subgroup analysis by sex revealed significant results in both subgroups (P value < 0.0001 and P value = 0.023 for males and females, respectively). Expression of CYP27B1 was up-regulated in cases vs. controls (Posterior beta = 0.415, P < 0.0001). Such pattern was also detected among males (P < 0.0001), but not females (P = 0.419). Similarly, MALAT1 and Linc00346 were up-regulated in total cases vs. controls (Posterior beta = 0.694, P < 0.0001 and Posterior beta = 0.4, P = 0.012, respectively) and in male cases compared with male controls (Posterior beta = 0.712, P < 0.0001 and Posterior beta = 0.41, P value = 0.038, respectively). Expression of VDR was up-regulated in total cases compared with controls (Posterior beta = 0.683, P value = 0.001). Finally, expression of Linc00511 was not different between groups. MALAT1, SNHG6, CYP27B1, VDR and Linc00346 had AUC values of 0.95, 0.94, 0.91, 0.85 and 0.83 in differentiation of male patients from controls, respectively.ConclusionThe current study suggests VDR-associated genes as possible markers for BD.

Increased MYD88 blood transcript in a mouse model of Alzheimer\u2019s disease

BackgroundNeuroinflammation plays a prominent role in Alzheimer’s disease (AD), both in pathogenesis and disease progression. It has been shown that TLR/MYD88 signaling is involved in the chronic low-grade sterile inflammation associated with AD. Several studies have evidenced high levels of MYD88 in the brain of patients and animal models of AD, but no study has assessed so far its levels in blood.MethodsIn this study we evaluated the blood mRNA levels of MYD88 in a mouse model of AD, and also the putative effect of Rivastigmine treatment on MYD88 expression. Twenty-eight transgenic APP/TAU mice (AT) and twenty-two control C57/BL6j mice (WT) were included in this study, out of which five transgenic AT and five WT mice were treated with Rivastigmine.ResultsIncreased MYD88 transcript in the whole blood from AT mice as compared to WT controls was found, which seems to increase in time due to disease progression and not to aging. This finding suggests that blood leukocytes are primed to develop TLR/MYD-mediated inflammatory processes. Moreover, results indicate that MYD88 blood levels were not modulated by the diseases-specific treatment with Rivastigmine.ConclusionsOur results suggest that MYD88 might be a promising blood biomarker to monitor AD progression.

Awardee Talk: Supplementing Omega-6 Fatty Acids to Beef Cows--An Alternative to Improve Overall Beef Production Efficiency.

Abstract Four experiments were conducted to evaluate: (1) effects of calcium salts of soybean oil (CSSO) supplementation to Bos taurus beef cows post-AI on conception rates and (2) on pregnancy establishment factors; (3) effects of CSSO supplementation to late gestating beef cows on the performance of the offspring; (4) the viability of utilizing low-moisture molasses-based blocks (LMB) as a delivery method for CSSO. In Exp. 1, 1,771 cows were divided into groups, and inseminated on d 0. After AI, groups received CSSO (n = 11), or prilled saturated fat (CON; n = 11) from d 0 to 21. Cows receiving CSSO had greater (P = 0.01) pregnancy rates. In Exp. 2, 90 cows housed in 18 pens were assigned to the same treatments and timed AI program from Exp. 1. On d 15, selected cows were assigned to conceptus collection. On d 20 blood was sampled for RNA extraction. CSSO supplementation increased (P = 0.05) mRNA expression of IFNT by the conceptus, and blood mRNA expression of ISGs. In Exp. 3, cows were assigned to receive: CSSO (n = 52) or CON (n = 52) during late gestation. CSSO cattle had greater (P ≤ 0.02) colostrum and plasma IgG; greater (P ≤ 0.05) expression of adipogenic and myogenic genes; required fewer microbial treatments for BRD (P = 0.05) and had greater longissimus muscle area compared to CON cohorts. In Exp. 4, 36 cows (n = 9 pens) were assigned to receive: 1) NOSUPP; 2) LMB, 24.7% CSSO; 3) CONC, hand-fed, 24.7% CSSO. Plasma FA were modified (P &amp;lt; 0.01) in CONC and LMB vs. NOSUPP cows. Collectively, these results present CSSO supplementation as a strategy to improve reproductive success in Bos taurus beef cows and productive performance of offspring born from supplemented dams. These results are associated to effects of linoleic acid and its ω-6 derivates. Additionally, the use of LMB seems to be a valid delivery method for CSSO supplementation, and consequently ω-6 FA, to beef cows.