Peripheral Blood RNA Research Articles

P-1850. Acute Babesia microti Infection in Humans Is Associated With Marked Changes In The Expression Of Peripheral Blood Coding And Noncoding RNA

Abstract Background Babesiosis is a potentially life-threatening disease transmitted by ticks. However, host and parasite determinants of human babesiosis are completely unknown. In the current study we evaluated the impact of B. microti infection, the primary cause of human babesiosis in the US, on the peripheral blood transcriptome. Figure 1. PCA of top 500 differentially expressed genes. PCA distinguishes Babesia patients at presentation from healthy controls. Methods Patients with confirmed B. microti acute infection were enrolled into a 1-year follow up cohort study at Stony Brook University Hospital, NY. Total RNA was isolated from blood using PaxGene RNA tubes and library preparation performed using Illumina's Stranded Total RNAPrep with Ribo-zero plus kit to remove human ribosomal RNA and globin RNA. 100 M RNA paired end reads were sequenced (2 x 151 bps). Low expression RNAs with less than 10 reads in 17 samples were filtered out prior to identification of differentially expressed genes (DEGs) (p< 0.05).Figure 2.Volcano Plot comparing differential gene and noncoding RNA expression between healthy controls and patients with babesiosis at presentation.Red circles represent genes whose expression are increased or decreased in patients with babesiosis at presentation compared to healthy controls. Blue circles represent genes whose expression are not significantly different. Results The peripheral blood transcriptome from 20 healthy controls was compared to 42 patients at presentation for B. microti infection and 1, 3, 6, 9 and 12 months after treatment for patients who completed the longitudinal protocol after the first visit. A total of 1860 genes or noncoding RNAs were differentially expressed in patients with babesiosis at presentation (V0) compared to healthy controls; 370 were decreased in patients with babesiosis and 1490 were increased (adj p < 0.01; expression fold change >+/- 1.) PCA analysis (Fig 1), and the Volcano Plot (Fig. 2) demonstrate that patients with babesiosis have a distinct peripheral blood gene expression profile compared to healthy control individuals. The heat map of DEGs in Fig. 3 shows the peripheral blood transcriptome in patients with babesiosis largely returned to baseline levels by 1-month post-treatment. The Qiagen IPA Graphical Summary based on DEGs is shown in Figure 4 and highlights activation of diverse immune-mediators, including TNF and interferon families, as well as growth factors, and vascularization pathways in response to B. microti infection.Figure 3.Heat map of differentially expressed genes of patients with babesiosis at presentation (0) and 1, 3, 6, 9 and 12 months post-treatment compared to healthy control individuals.Gene expression in patients with babesiosis is markedly different at presentation but is similar to levels for healthy controls by 1 month post-treatment. Conclusion Infection with B. microti results in marked changes in expression of genes and noncoding RNA in peripheral blood that largely returns to baseline by 1-month post-treatment.Figure 4:Qiagen IPA Graphical Summary of pathways likely increased (orange) or decreased (blue) in patients presenting with babesiosis. Disclosures Paul Arnaboldi, PhD, Biopeptides, Corp: Patent Holder|Biopeptides, Corp: Employee|DiaSorin: Advisor/Consultant

P1002 Changes in circulating lymphocyte subsets and CCR9 transcripts as mechanistic biomarkers of the small molecule α4β7 inhibitor MORF-057 in patients with Ulcerative Colitis

Abstract Background MORF-057 is an orally administered, selective, and potent small molecule inhibitor of the cell surface receptor integrin a4b7. Upon binding to the a4b7 heterodimer, MORF-057 blocks the interaction of this integrin with the endothelial ligand mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) which, in turn, prevents the extravasation of circulating lymphocytes into the intestinal mucosa. In this study, the pharmacodynamic effects of MORF-057 on immune cell trafficking were evaluated in UC patients using a flow cytometry-based assay and by assessing the expression of CCR9 gene in blood. Methods As part of the induction phase of an open-label, single-arm, Phase 2a study (NCT05291689) the efficacy, safety, and tolerability of MORF-057 in 35 patients with moderately to severely active UC disease were evaluated. Patients were treated with MORF-057 at a dose of 100 mg BID orally for an induction period of 12 weeks followed by a 40-week maintenance period. Blood was collected for peripheral blood mononuclear cell (PBMC) isolation and RNA extraction at baseline and post-treatment at Week 2, 6, 12, 20, 28, 36, 44, and 52. Using flow cytometry, levels of CD4+β7+ naïve, CD4+β7+ central memory (TCM), CD4+β7 Hi effector memory (TEM) T cells, and CD19+β7+ switched memory B cells (BSM) subpopulations were quantified as a percentage of the parent populations CD4 naive, TCM, TEM and BSM, respectively, then normalized to pre-treatment baseline. Expression of CCR9 mRNA in the blood was quantified as a fold ratio relative to baseline for the same timepoints. Statistical analyses were performed using the Wilcoxon Signed Rank test. Results The percentages of circulating lymphocyte subsets CD4+β7 Hi TEM cells, CD4+β7+ TCM and CD19+β7+ BSM were significantly elevated relative to baseline as early as Week 2 and up to Week 52 (Table 1). These changes were not observed in the overall lymphocyte counts nor in the CD4+β7+Naïve T cells (except at Week 52). The baseline-normalized expression levels of CCR9 at Weeks 12 and 52 were 1.6 [0.6] (mean [SD]) and 1.7 [0.8], respectively (Figure 1). Conclusion Patients receiving MORF-057 for a period of up to 52 weeks achieved statistically significant increases in specific β7+ lymphocyte subsets expressing ITGβ7. Elevated levels of the CCR9 transcript were observed in blood over the course of treatment. Effects observed were consistent with those reported for patients receiving biologic inhibitors of the same pathway as well as in healthy volunteers receiving MORF-0571,2. These changes in circulating lymphocyte populations and CCR9 transcript levels may be used as mechanistic biomarkers for MORF-057 in UC patients.

Role of TLR7 in the immunopathogenesis of psoriasis and psoriatic arthritis

BACKGROUND: Toll-like receptor 7 (TLR7) plays a significant role in the development of inflammation in psoriasis. However, there are very few data on the role of different polymorphic markers of the TLR7 gene in psoriasis and psoriatic arthritis. AIM: Study of the role of polymorphic marker rs179009 of TLR7 gene in patients with severe psoriasis. MATERIALS AND METHODS: The study was conducted during the period of 2016–2024. The analysis of innate immunity indicators in the study group was performed by obtaining RNA in peripheral venous blood and polymorphisms of the gene recognizing marker rs179009 of the TLR7 receptor were investigated by polymerase chain reaction with TaqMan probes. To assess the area and severity of psoriatic lesions of the skin process, we used a standardised method of assessment — determination of the PASI index. RESULTS: The main group consisted of 168 patients (100%) with psoriasis. Of these, 45 (26.8%) were women and 123 men (73.2%). The average age of the patients was 54.0±14.0 years. The mean duration of psoriasis course was 11.8±0.6 years. The mean psoriasis area and severity index (PASI) value was 17.7±7.2. When analyzing the genotypes of polymorphic marker rs179009 in the TLR7 gene in patients with psoriasis of different severity, it was revealed that heterozygote CT was significantly more frequent in patients with mild psoriasis, and homozygote CC and homozygote TT were registered at PASI 10 (p 0.05). The C allele of the studied marker was significantly more frequent in patients with late psoriasis debut and late development of psoriatic arthritis (p 0.01). In contrast, the T allele was significantly more frequent in patients with early debut of psoriasis and psoriatic arthritis (p 0.01). When analyzing the genotypes of polymorphic marker rs179009 in the TLR7 gene, the occurrence of homozygous CC and TT genotypes was also found to have statistically significant differences. The CC genotype was more frequently registered in patients with late onset of psoriasis and late onset of arthritis (p 0.05). In contrast, the TT genotype was significantly more frequently registered in patients with the debut of skin and joint process before 40 years of age (p 0.05). CONCLUSION: In our study it was found that homozygous carriage of CC and TT genotypes of polymorphic marker rs179009 in the TLR7 gene predisposes to a more severe course of the skin process in psoriasis. Also, the presence in the patient of allele C or homozygote CC of marker rs179009 in the TLR7 gene is a predictor of late onset of the skin process and the development of arthritis. While the presence of the T allele or TT homozygote statistically significantly predisposes to early onset of psoriasis and early onset of joint symptoms with the development of psoriatic arthritis.

Gain of bipolar disorder-related lncRNA AP1AR-DT in mice induces depressive and anxiety-like behaviors by reducing Negr1-mediated excitatory synaptic transmission

BackgroundBipolar disorder is a complex polygenic disorder that is characterized by recurrent episodes of depression and mania, the heterogeneity of which is likely complicated by epigenetic modifications that remain to be elucidated.MethodsWe performed transcriptomic analysis of peripheral blood RNA from monozygotic (MZ) twins discordant for bipolar disorder to identify disease-associated differentially expressed long noncoding RNAs (DE-lncRNAs), which were further validated in the PsychENCODE brain RNA-seq dataset. We then performed behavioral tests, electrophysiological assays, chromatin immunoprecipitation, and PCR to investigate the function of DE-lncRNAs in the mouse and cell models. Statistical analyses were performed using GraphPad Prism 9.0 or SPSS.ResultsWe identified a bipolar disorder-associated upregulated long non-coding RNA (lncRNA), AP1AR-DT. We observed that overexpression of AP1AR-DT in the mouse medial prefrontal cortex (mPFC) resulted in a reduction of both the total spine density and the spontaneous excitatory postsynaptic current (sEPSC) frequency of mPFC neurons as well as depressive and anxiety-like behaviors. A combination of the results of brain transcriptome analysis of AP1AR-DT overexpressing mice brains with the known genes associated with bipolar disorder revealed that NEGR1, which encodes neuronal growth regulator 1, is one of the AP1AR-DT targets and is reduced in vivo upon gain of AP1AR-DT in mice. We further demonstrated that overexpression of recombinant Negr1 in the mPFC neurons of AP1AR-DTOE mice ameliorates depressive and anxiety-like behaviors and normalizes the reduced excitatory synaptic transmission induced by the gain of AP1AR-DT. We finally identified that AP1AR-DT reduces NEGR1 expression by competing for the transcriptional activator NRF1 in the overlapping binding site of the NEGR1 promoter region.ConclusionsThe epigenetic and pathophysiological mechanism linking AP1AR-DT to the modulation of depressive and anxiety-like behaviors and excitatory synaptic function provides etiological implications for bipolar disorder.

Characterizing Non-T2 Asthma: Key Pathways and Molecular Implications Indicative of Attenuated Th2 Response.

Non-Type 2 (non-T2) asthma is characterized by a lack of allergic sensitization and normal to low total IgE levels. We aimed to explore molecular mechanisms and pathways differentiating non-T2 from T2-high pediatric asthma. We analyzed peripheral blood RNA samples from 11 non-T2 and 17 T2-high pediatric asthma patients using bulk RNA sequencing. Differentially expressed genes (DEGs) were identified, followed by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, and Protein-Protein Interaction (PPI) network construction. Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) were employed to explore significance of these DEGs. We utilized independent public datasets GSE145505 to validate our findings. We investigated Th cytokine profiles in an independent cohort of pediatric patients with non-T2 asthma (n = 38) and T2-high asthma (n = 64). We demonstrated that the total serum IgE levels of children with non-T2 asthma (128.4 ± 159.5IU/mL) was significantly lower than that of those with T2-high asthma (405.8 ± 252.1IU/mL). Our analysis revealed 136 DEGs distinguishing non-T2 from T2-high asthma. IPA identified predicted inhibition of IgE-FcεRI signaling pathways in non-T2 asthma. Our DEG data showed the expression of IGHV4-39, IGLV1-40, IGLV1-47, IGLV1-44, IGHV1-69, IGLV6-57, IGLV3-19, IGLV3-1, and IGLC7 were downregulated in our non-T2 asthma patient. The non-T2 group exhibited significantly higher concentrations of IL-2, IFN-γ, IL-6, and IL-17A compared to the T2-high group. Our integrated analysis differentiated non-T2 from T2-high asthma by revealing downregulation of specific immunoglobulin genes influencing FcεRI signaling, elevated Th1 cytokines and Th17 cytokines might affect IgE associated sensitization and alter Th2 allergic response.

Expression of interferon-stimulated genes, but not polymorphisms in the interferon α/β receptor 2 gene, is associated with coronavirus disease 2019 mortality

Excessive inflammatory response is a hallmark of severe COVID-19. This study investigated the associations between interferon-stimulated genes (ISGs) expression, genetic variation in the interferon α/β receptor 2 (IFNAR2) gene, and COVID-19 mortality.We investigated 67 patients with moderate-to-severe COVID-19. Of them, 22 patients (32.8 %) died because of COVID-19. We examined the expression of ISGs in total RNA of peripheral whole blood. We observed a significant increase in the expression of all ISGs examined in non-surviving patients, indicating a heightened interferon type I signaling activation in non-survived patients. Subsequently, we analyzed whether the increase in ISGs expression was correlated with polymorphism within the IFNAR2 gene. Intriguingly, no significant association was observed between IFNAR2 gene polymorphism and COVID-19 mortality. Similarly, no association was noted between the IFNAR2 and ISGs expression levels.Overall, our data showed that higher ISGs expression, which presumably indicates heightened interferon type I activation, is associated with COVID-19 mortality.

Divergent transcriptomic profiles in depressed individuals with hyper- and hypophagia implicating inflammatory status

BackgroundMajor Depressive Disorder (MDD) is a heterogenous and etiologically complex disease often presenting with divergent appetitive phenotypes including Hyperphagic MDD (characterized by an increased appetite) and Hypophagic MDD (characterized by a decrease in appetite) which are closely related to comorbidities, including cardiometabolic disorders. Hyperphagia is associated with atypical depression, decreased stress-hormone signaling, a pro-inflammatory status, hypersomnia, and poorer clinical outcomes. Yet, our understanding of associated biological correlates of Hyperphagic and Hypophagic MDD remain fragmented. MethodsWe performed an exploratory study on peripheral blood RNA profiling using bulk RNAseq in unmedicated individuals with Hyperphagic and Hypophagic MDD (n = 7 and n = 13, respectively). ResultsAt baseline, we discovered an increased expression of TADA2B in hyperphagic MDD with the significant enrichment of 72 gene ontology pathways mainly related to inflammation. In addition, we used the Maastricht Acute Stress Task to uncover stress-related transcriptomic profiles in Hyper- and Hypophagic MDD and discovered the upregulation of CCDC196 and the downregulation of SPATA33 in hyperphagic MDD. Gene ontology enrichment analysis after stress exposure showed pathways related to ribosomal activity. Limitations: The present findings are tempered primarily by the limited sample size, which requires independent replication of this exploratory study. However, stringent methods controlling for false positive findings mitigate the risk associated with sample size limitations. DiscussionLimitations notwithstanding, findings suggest that hyper- and hypophagic MDD is associated with divergent RNA expression profiles in peripheral blood that are amplified by exposure to a controlled stress test. Our findings in a well-controlled study provide evidence for peripheral markers of a relevant endophenotype of MDD.

Dl-3-n-butylphthalide improves stroke outcomes after focal ischemic stroke in mouse model by inhibiting the pyroptosis-regulated cell death and ameliorating neuroinflammation

Recent studies have highlighted the involvement of pyroptosis-mediated cell death and neuroinflammation in ischemic stroke (IS) pathogenesis. DL-3-n-butylphthalide (NBP), a synthesized compound based on an extract from seeds of Apium graveolens, possesses a broad range of biological effects. However, the efficacy and the underlying mechanisms of NBP in IS remain contentious. Herein, we investigated the therapeutic effects of NBP and elucidated its potential mechanisms in neuronal cell pyroptosis and microglia inflammatory responses. Adult male mice underwent permanent distal middle cerebral artery occlusion (dMCAO), followed by daily oral gavage of NBP (80 mg/kg) for 1, 7, or 21 consecutive days. Gene Expression Omnibus (GEO) dataset of IS patients peripheral blood RNA sequencing was analyzed to identify differentially expressed pyroptosis-related genes (PRGs) during the ischemic process. Our results suggested that NBP treatment effectively alleviated brain ischemic damage, resulting in decreased neurological deficit scores, reduced infarct volume, and improved neurological and behavioral functions. RNA sequence data from human unveiled upregulated PRGs in IS. Subsequently, we observed that NBP downregulated pyroptosis-associated markers at days 7 and 21 post-modeling, at both the protein and mRNA levels. Additionally, NBP suppressed the co-localization of pyroptosis markers with neuronal cells to variable degrees and simultaneously mitigated the accumulation of activated microglia. Overall, our data provide novel evidence that NBP treatment significantly attenuates ischemic brain damage and promotes recovery of neurological function in the early and recovery phases after IS, probably by negatively regulating the pyroptosis cell death of neuronal cells and inhibiting toxic neuroinflammation in the central nervous system.

Extraction of high quality and high yield RNA from frozen EDTA blood

Peripheral blood RNA profiling, which can reveal systemic changes in gene expression and immune responses to disease onset and progression, is a powerful tool for diagnosis and biomarker discovery. This technique usually requires high quality RNA, which is only obtainable from fresh blood, or frozen blood that has been collected in special RNA-stabilisation systems. The current study aimed to develop a novel protocol to extract high quality RNA from frozen blood that had been collected in the conventional EDTA tubes. We determined that thawing EDTA blood in the presence of cell lysis/RNA stabilisation buffers (Paxgene or Nucleospin) significantly improved RNA quality (RIN) from below 5 to above 7, which to date has not been shown possible. The EDTA-Nucleospin protocol resulted in 5 times higher yield than the EDTA-Paxgene-PreAnalytix method. The average RIN and mRNA expression levels of five different genes including 18 s, ACTB, MCP1, TNFa and TXNIP using this protocol were also indifferent to those from Paxgene blood, suggesting similar RNA quality and blood transcriptome. Moreover, the protocol allows DNA to be extracted simultaneously. In conclusion, we have developed a practical and efficient protocol to extract high quality, high yield RNA from frozen EDTA blood.

Decreased telomere length in a subgroup of young individuals with bipolar disorders: replication in the FACE-BD cohort and association with the shelterin component POT1

IntroductionA 10-15 years decrease in life expectancy has been observed in individuals with bipolar disorder (BD) and has been associated with premature cellular aging, but mechanisms involved remain unclear. Our team recently identified a subgroup of young individuals with prematurely shortened telomere length (TL).ObjectivesThe aims of the present study were to replicate this observation in a larger sample and to analyze the expression levels of genes associated with age or TL in a subsample of these individuals.Methods TL was measured by qPCR using peripheral blood DNA from 542 individuals with BD. Clustering analyzes were performed with age and TL as classification variables to identify similar groups.Gene expression of 29 genes, including 20 associated with age and 9 with TL, was analyzed by RT-qPCR using peripheral blood RNA in a subgroup of 129 individuals. Gene expressions were compared between groups obtained from the previous clustering analyzes by Kruskal-Wallis and Mann-Whitney tests.ResultsClustering analyzes identified 3 subgroups and replicated the clustering previously described: a subgroup of aged individuals with a low TL (mean age : 51.73 years ; mean TL : 2), a subgroup of young individuals with a high TL (mean age : 29.02 years ; mean TL : 4.36) and a subgroup of young individuals but with a low TL (mean age : 29.64 years ; mean TL : 1.96). None of the tested clinical variables were significantly associated with this subgroup.Furthermore, gene expression level analyzes showed that only POT1 expression was different between the two subgroups of young individuals, with a downregulation of POT1 expression in the subgroup with a lower TL level. POT1 is a protein involved in the maintenance of TL. POT1 binds to another protein TPP1 allowing the recruitment of telomerase, the enzyme which extends TL. Our hypothesis is that in the subgroup presenting a lower POT1 expression, the POT1-TPP1 complex cannot form and thus prevents telomerase recruitment and TL elongation.ConclusionsThis study confirms, on a larger sample, the existence of a subgroup of young individuals with BD presenting accelerated cellular aging. The observed decrease of POT1 expression level suggests a newly described cellular mechanism in individuals with BD, that may contribute to telomere shortening.Disclosure of InterestNone Declared

Abstract 2661: Identification of a novel morphology of tertiary lymphoid structure in patients with hepatocellular carcinoma treated with neoadjuvant immune checkpoint blockade

Abstract Background: Tertiary lymphoid structures (TLS) are ectopic lymphoid follicles that arise in non-lymphoid tissue. TLS may contribute to response to immune checkpoint blockade (ICB) in solid tumors, but understanding of the life cycle of these structures, particularly the circumstances of their resolution and the functional contribution of this stage to the adaptive immune response, remains incomplete. Methods: We employed a multi-omics approach to evaluate TLS in the tumors of patients with hepatocellular carcinoma (HCC) treated with neoadjuvant ICB (n = 19) and untreated controls (n = 14). TLS density was assessed by immunohistochemistry and correlated with pathologic response, relapse free survival, and overall survival. Imaging mass cytometry was used to characterize distinct TLS morphologies in areas of tumor regression bed and viable tumor. Individual TLS from treated tumors were microdissected and characterized by T cell receptor (TCR) and immunoglobulin heavy chain (IGH) sequencing, and TCR clonotypes identified in TLS were further characterized by comparison against matched single cell RNA and TCR sequencing of peripheral blood and TIL. Results: Intratumoral TLS density was significantly increased in HCC tumors treated with neoadjuvant ICB compared to untreated controls (p = 0.05). Tumors with major or complete pathologic response to treatment had significantly higher intratumoral TLS density compared to tumors with partial pathologic response (p = 0.000246) and non-response (p = 0.0129). In the treatment group, patients with tumors in the top tertile of intratumoral TLS density had significantly longer relapse free survival (p = 0.021 by log-rank test). In the regression bed of treated tumors, an involuted TLS morphology was identified which was notable for dispersion of the B cell germinal center, retention of a T cell zone enriched for T-DC interactions, and increased expression of markers of T cell memory. In a subset of patients, immune repertoire sequencing demonstrated increased TCRβ clonality and BCR somatic hypermutation at involuted TLS. Highly expanded T cell clonotypes identified in TLS were found in the GZMK+ and GZMB+ CD8 T effector memory clusters in matched single cell data. Conclusions: These data suggest that in HCC tumors treated with neoadjuvant ICB, treatment may induce formation of intratumoral TLS, which are associated with superior pathologic response and relapse free survival. An involuted TLS morphology is identified in tumor regression bed with features consistent with dissolution of the B cell germinal center, persistent antigen presentation to T cells, and increased T cell memory formation. Taken together, these findings suggest a potential role for late-stage TLS in supporting consolidation of the intratumoral T cell repertoire after elimination of viable tumor. Citation Format: Daniel H. Shu, Won Jin Ho, Luciane T. Kagohara, Alexander Girgis, Sarah M. Shin, Ludmila Danilova, Jae W. Lee, Dimitrios N. N. Sidiropoulos, Sarah Mitchell, Kabeer Munjal, Kathryn Howe, Kayla J. Bendinelli, Hanfei Qi, Guanglan Mo, Janelle Montagne, Tamara Y. Lopez-Vidal, Qingfeng Zhu, Amanda L. Huff, Xuan Yuan, Alexei Hernandez, Erin M. Coyne, Neeha Zaidi, Daniel J. Zabransky, Logan L. Engle, Aleksandra Ogurtsova, Marina Baretti, Daniel Laheru, Jennifer N. Durham, Hao Wang, Robert Anders, Elizabeth M. Jaffee, Elana J. Fertig, Mark Yarchoan. Identification of a novel morphology of tertiary lymphoid structure in patients with hepatocellular carcinoma treated with neoadjuvant immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2661.

N6-methyladenosine levels in peripheral blood RNA: a potential diagnostic biomarker for colorectal cancer

BackgroundN6-methyladenosine (m6A) is dysregulated in various cancers, including colorectal cancer (CRC). Herein, we assess the diagnostic potential of peripheral blood (PB) m6A levels in CRC.MethodsWe collected PB from healthy controls (HCs) and patients with CRC, analyzed PB RNA m6A levels and the expression of m6A-related demethylase genes FTO and ALKBH5, cocultured CRC cells with PB mononuclear cells (PBMCs), and constructed an MC38 cancer model.ResultsPB RNA m6A levels were higher in the CRC than that in HCs. The area under the curve (AUC) of m6A levels (0.886) in the CRC was significantly larger compared with carbohydrate antigen 199 (CA199; 0.666) and carcinoembryonic antigen (CEA; 0.834). The combination of CEA and CA199 with PB RNA m6A led to an increase in the AUC (0.935). Compared with HCs, the expression of FTO and ALKBH5 was decreased in the CRC. After coculturing with CRC cells, the PBMCs RNA m6A were significantly increased, whereas the expression of FTO and ALKBH5 decreased. Furthermore, m6A RNA levels in the PB of MC38 cancer models were upregulated, whereas the expression of FTO and ALKBH5 decreased.ConclusionsPB RNA m6A levels are a potential diagnostic biomarker for patients with CRC.

Peripheral blood RNA biomarkers can predict lesion severity in degenerative cervical myelopathy.

JOURNAL/nrgr/04.03/01300535-202506000-00027/figure1/v/2024-08-05T133530Z/r/image-tiff Degenerative cervical myelopathy is a common cause of spinal cord injury, with longer symptom duration and higher myelopathy severity indicating a worse prognosis. While numerous studies have investigated serological biomarkers for acute spinal cord injury, few studies have explored such biomarkers for diagnosing degenerative cervical myelopathy. This study involved 30 patients with degenerative cervical myelopathy (51.3 ± 7.3 years old, 12 women and 18 men), seven healthy controls (25.7 ± 1.7 years old, one woman and six men), and nine patients with cervical spondylotic radiculopathy (51.9 ± 8.6 years old, three women and six men). Analysis of blood samples from the three groups showed clear differences in transcriptomic characteristics. Enrichment analysis identified 128 differentially expressed genes that were enriched in patients with neurological disabilities. Using least absolute shrinkage and selection operator analysis, we constructed a five-gene model (TBCD, TPM2, PNKD, EIF4G2, and AP5Z1) to diagnose degenerative cervical myelopathy with an accuracy of 93.5%. One-gene models (TCAP and SDHA) identified mild and severe degenerative cervical myelopathy with accuracies of 83.3% and 76.7%, respectively. Signatures of two immune cell types (memory B cells and memory-activated CD4+ T cells) predicted levels of lesions in degenerative cervical myelopathy with 80% accuracy. Our results suggest that peripheral blood RNA biomarkers could be used to predict lesion severity in degenerative cervical myelopathy.

Investigating The Expression Changes of miR-93-3p, NFATc1 and NFATc3 Genes in Peripheral Blood Mononuclear Cells (PBMC) of Breast Cancer Patients

Background & Objectives: Breast cancer is the most lethal malignancy in women. miRNAs function as epigenetic regulators and contribute to the pathogenesis of breast cancer. Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and 3 (NFATc3), are targeted by microRNA-93 (miR93). This study aims to evaluate the expression of these genes in peripheral blood mononuclear cells (PBMCs) of healthy women and women with breast cancer Materials & Methods: In this cross-sectional study, blood samples were collected from 20 healthy women and 20 women with early-stage breast cancer. After isolating peripheral blood mononuclear cells (PBMCs), RNA extraction and cDNA synthesis were performed. The expression of the desired genes was then examined by Real-Time Polymerase Chain Reaction (RT-PCR). Statistical analysis was conducted. A p-value less than 0.05 was considered statistically significant, and Student’s t-test was used to evaluate the relative changes in gene expression. Results: The results demonstrated that the expression of NFATc1 and NFATc3 genes in peripheral blood mononuclear cells (PBMCs) of breast cancer patients was significantly reduced compared to their expression in healthy individuals. Conversely, the expression of the miR-93-3p gene was significantly lower in healthy women than in breast cancer patients (p < 0.05). Conclusions: This study investigated the expression of miR-93-3p and its downstream targets, the NFATc1 and NFATc3 genes, for the first time in peripheral blood mononuclear cells. The expression levels were shown to be significantly different in patients with breast cancer compared to healthy women.

Computational analysis of peripheral blood RNA sequencing data unravels disrupted immune patterns in Alzheimer's disease.

The central nervous system (CNS) and the immune system collectively coordinate cellular functionalities, sharing common developmental mechanisms. Immunity-related molecules exert an influence on brain development, challenging the conventional view of the brain as immune-privileged. Chronic inflammation emerges as a key player in the pathophysiology of Alzheimer's disease (AD), with increased stress contributing to the disease progression and potentially exacerbating existing symptoms. In this study, the most significant gene signatures from selected RNA-sequencing (RNA-seq) data from AD patients and healthy individuals were obtained and a functional analysis and biological interpretation was conducted, including network and pathway enrichment analysis. Important evidence was reported, such as enrichment in immune system responses and antigen processes, as well as positive regulation of T-cell mediated cytotoxicity and endogenous and exogenous peptide antigen, thus indicating neuroinflammation and immune response participation in disease progression. These findings suggest a disturbance in the immune infiltration of the peripheral immune environment, providing new challenges to explore key biological processes from a molecular perspective that strongly participate in AD development.

Development and Optimization of a Subtraction-Normalized Immunocyte Profiling Signature for Prostate Cancer Active Surveillance Risk Stratification.

Less invasive decision support tools are desperately needed to identify occult high-risk disease in men with prostate cancer (PCa) on active surveillance (AS). For a variety of reasons, many men on AS with low- or intermediate-risk disease forgo the necessary repeat surveillance biopsies needed to identify potentially higher-risk PCa. Here, we describe the development of a blood-based immunocyte transcriptomic signature to identify men harboring occult aggressive PCa. We then validate it on a biopsy-positive population with the goal of identifying men who should not be on AS and confirm those men with indolent disease who can safely remain on AS. This model uses subtraction-normalized immunocyte transcriptomic profiles to risk-stratify men with PCa who could be candidates for AS. Men were eligible for enrollment in the study if they were determined by their physician to have a risk profile that warranted prostate biopsy. Both training (n = 1017) and validation cohort (n = 1198) populations had blood samples drawn coincident to their prostate biopsy. Purified CD2+ and CD14+ immune cells were obtained from peripheral blood mononuclear cells, and RNA was extracted and sequenced. To avoid overfitting and unnecessary complexity, a regularized regression model was built on the training cohort to predict PCa aggressiveness based on the National Comprehensive Cancer Network PCa guidelines. This model was then validated on an independent cohort of biopsy-positive men only, using National Comprehensive Cancer Network unfavorable intermediate risk and worse as an aggressiveness outcome, identifying patients who were not appropriate for AS. The best final model for the AS setting was obtained by combining an immunocyte transcriptomic profile based on 2 cell types with PSA density and age, reaching an AUC of 0.73 (95% CI: 0.69-0.77). The model significantly outperforms (P < .001) PSA density as a biomarker, which has an AUC of 0.69 (95% CI: 0.65-0.73). This model yields an individualized patient risk score with 90% negative predictive value and 50% positive predictive value. While further validation in an intended-use cohort is needed, the immunocyte transcriptomic model offers a promising tool for risk stratification of individual patients who are being considered for AS.

Reclassification of variants of tumor suppressor genes based on Sanger RNA sequencing without NMD inhibition.

Introduction: RNA sequence analysis can be effectively used to identify aberrant splicing, and tumor suppressor genes are adequate targets considering their loss-of-function mechanisms. Sanger sequencing is the simplest method for RNA sequence analysis; however, because of its insufficient sensitivity in cases with nonsense-mediated mRNA decay (NMD), the use of cultured specimens with NMD inhibition has been recommended, hindering its wide adoption. Method: The results of Sanger sequencing of peripheral blood RNA without NMD inhibition performed on potential splicing variants of tumor suppressor genes were retrospectively reviewed. For negative cases, in which no change was identified in the transcript, the possibility of false negativity caused by NMD was assessed through a review of the up-to-date literature. Results: Eleven potential splice variants of various tumor suppressor genes were reviewed. Six variants were classified as pathogenic or likely pathogenic based on the nullifying effect identified by Sanger RNA sequencing. Four variants remained as variants of uncertain significance because of identified in-frame changes or normal expression of both alleles. The result of one variant was suspected to be a false negative caused by NMD after reviewing a recent study that reported the same variant as causing a nullifying effect on the affected transcript. Conclusion: Although RNA changes found in the majority of cases were expected to undergo NMD by canonical rules, most cases (10/11) were interpretable by Sanger RNA sequencing without NMD inhibition due to incomplete NMD efficiency or allele-specific expression despite highly efficient NMD.

Effect of vitamin D binding protein gene polymorphism on susceptibility and prognosis of severe acute pancreatitis

To investigate the effect of vitamin D binding protein (DBP) gene polymorphism on susceptibility and prognosis of severe acute pancreatitis (SAP). A prospective study was conducted. Eighty-three patients with SAP who were admitted to the department of general surgery of Tianjin Fifth Central Hospital from March 2018 to March 2021 were selected as the research objects, and 83 healthy people in the same period were selected as controls. Peripheral blood RNA was extracted and reverse transcribed into cDNA, and the genotype and allele frequency of DBP gene rs7041 locus were detected by fluorescence quantitative analyzer. Hardy-Weinberg equilibrium was used to test the genetic balance. On the day of admission, serum C-reactive protein (CRP) level was detected by scattering immunoturbidimetry, serum procalcitonin (PCT) level was detected by electrochemiluminescence, serum DBP level was detected by enzyme-linked immunosorbent assay (ELISA), and neutrophil to lymphocyte ratio (NLR) was calculated automatically by the instrument. The length of intensive care unit (ICU) stay, the length of hospital stay and prognosis during hospitalization of patients were statistically analyzed. Multivariate Logistic regression analysis was used to screen the influencing factors of SAP occurrence. The results of Hardy-Weinberg equilibrium test showed that the distribution of gene polymorphisms in the two groups of subjects conformed to the law of genetic equilibrium. The frequencies of TT genotype and T allele of DBP gene rs7041 locus in the patients of SAP group were significantly higher than those in the healthy control group [TT genotype: 34.94% (29/83) vs. 9.64% (8/83), T allele: 55.42% (92/166) vs. 38.55% (64/166), both P < 0.01], and the frequency of GT genotype was significantly lower than that in the healthy control group [40.96% (34/83) vs. 57.83% (48/83), P < 0.05]. There was no significant difference in the frequency of GG genotype between the healthy control group and SAP group [32.53% (27/83) vs. 24.10% (20/83), P > 0.05]. Further multivariate Logistic regression analysis showed that TT genotype [odds ratio (OR) = 2.831, 95% confidence interval (95%CI) was 1.582-5.067, P < 0.001] and T allele (OR = 2.533, 95%CI was 1.435-4.472, P < 0.001) of DBP gene rs7041 locus were independent risk factors for SAP in healthy people, while GT genotype was a protective factor for SAP (OR = 0.353, 95%CI was 0.143-0.868, P = 0.041). The levels of CRP, PCT, NLR and DBP in patients with TT genotype of DBP gene rs7041 locus were significantly higher than those in patients with GG/GT genotype on the day of admission in SAP group [CRP (mg/L): 43.25±13.25 vs. 31.86±12.83, PCT (μg/L): 1.53±0.24 vs. 1.21±0.20, NLR: 3.15±0.53 vs. 2.71±0.48, DBP (μg/L): 87.78±19.64 vs. 70.58±18.67, all P < 0.01]. The length of ICU stay in patients with TT genotype of DBP gene rs7041 locus in SAP group was significantly longer than that in patients with GG/GT genotype (days: 11.35±1.58 vs. 9.71±1.35, P < 0.01). The length of hospital stay of patients with TT genotype was longer than that of patients with GG/GT genotype (days: 23.41±3.64 vs. 23.17±3.57), and the in-hospital mortality was higher than that of patients with GG/GT genotype [34.48% (10/29) vs. 29.63% (16/54)], but the difference was not statistically significant (both P > 0.05). The risk of SAP was significantly increased in patients with TT genotype of rs7041 locus of DBP gene, and the mechanism may be related to the increase of DBP expression. And carrying the TT genotype will prolong the ICU hospitalization time of SAP patients, but the effect on prognosis is not obvious.

Investigation of LncRNA PVT1 and MiR-21-5p Expression as Promising Novel Biomarkers for Autism Spectrum Disorder.

The characteristics of ncRNA in children with autism spectrum disorder (ASD) were observed to disclose a theoretical basis for further research on molecular markers for early warning of ASD. Children with ASD and normal control children were recruited to collect peripheral blood RNA samples. The concentration of PVT1 and miR-21-5p was quantitatively analyzed by qRT-PCR. Pearson correlation coefficient method was used to evaluate the link between PVT1 level and miR-21-5p level of the children. Receiver operating characteristic (ROC) curves were applied to reckon the predictive value of PVT1, miR-21-5p, and their combination in ASD. The interconnection of PVT1 with miR-21-5p was represented by luciferase reporter assay. The targeted genes of miR-21-5p were predicted. The enrichment and protein interaction analysis of these genes was carried out to find the important core genes and analyze their value in ASD. In the disease group, the level of PVT1 was downregulated, while the content of miR-21-5p was upregulated. The expression level of serum miR-21-5p was negatively correlated with the level of PVT1. Luciferase reporter gene assay documented that PVT1 directly targeted miR-21-5p. ROC curve showed that PVT1, miR-21-5p, and their combination showed clinical value for disease diagnosis. The functional enrichment analysis showed that the targets of miR-21-5p participated in ASD by regulating related functions and pathways. Reduced expression of PVT1 and raised miR-21-5p were good diagnostic markers for ASD, which would provide a basis for effective prevention, early diagnosis, and early intervention of ASD.

Role of circ-FOXO3 and miR-23a in radiosensitivity of breast cancer.

Identifying the radiosensitivity of cells before radiotherapy (RT) in breast cancer (BC) patients allows appropriate switching between routinely used treatment regimens and reduces adverse side effects in exposed patients. In this study, blood was collected from 60 women diagnosed with Invasive Ductal Carcinoma (IDC) BC and 20 healthy women. To predict cellular radiosensitivity, a standard G2-chromosomal assay was performed. From these 60 samples, 20 BC patients were found to be radiosensitive based on the G2 assay. Therefore, molecular studies were finally performed on two equal groups (20 samples each) of patients with and without cellular radiosensitivity. QPCR was performed to examine the expression levels of circ-FOXO3 and miR-23a in peripheral blood mononuclear cells (PBMCs) and RNA sensitivity and specificity were determined by plotting Receiver Operating Characteristic (ROC) curves. Binary logistic regression was performed to identify RNA involvement in BC and cellular radiosensitivity (CR) in BC patients. Meanwhile, qPCR was used to compare differential RNA expression in the radiosensitive MCF-7 and radioresistant MDA-MB-231 cell lines. An annexin -V FITC/PI binding assay was used to measure cell apoptosis 24 and 48h after 2Gy, 4Gy, and 8Gy gamma-irradiation. Results indicated that circ-FOXO3 was downregulated and miR-23a was upregulated in BC patients. RNA expression levels were directly associated with CR. Cell line results showed that circ-FOXO3 overexpression induced apoptosis in the MCF-7 cell line and miR-23a overexpression inhibited apoptosis in the MDA-MB-231 cell line. Evaluation of the ROC curves revealed that both RNAs had acceptable specificity and sensitivity in predicting CR in BC patients. Binary logistic regression showed that both RNAs were also successful in predicting breast cancer. Although only circ-FOXO3 has been shown to predict CR in BC patients, circ-FOXO3 may function as a tumor suppressor and miR-23a may function as oncomiR in BC. Circ-FOXO3 and miR-23a may be promising potential biomarkers for BC prediction. Furthermore, Circ-FOXO3 could be a potential biomarker for predicting CR in BC patients.